Phospholinked Dye Analogs with an Amino Acid Linker

ABSTRACT

In various embodiments, the present invention provides fluorescent dyes that are linked to another species through an amino acid or peptide linker. In an exemplary embodiment, the dye is linked to a polyphosphate nucleic acid through an amino acid or peptide linker. These conjugates find use in single molecule DNA sequencing and other applications. In various embodiments, the dye moiety is a cyanine dye. Cyanine dyes that are highly charged, such as those including multiple sulfonate, alkylsulfonate, carboxylate and/or alkylcarboxylate moieties are examples of cyanine dyes of use in the compounds of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.61/377,004, filed on Aug. 25, 2010, 61/377,022, filed on Aug. 25, 2010,61/377,031, filed on Aug. 25, 2010, 61/377,038, filed on Aug. 25, 2010,and 61/377,048, filed on Aug. 25, 2010 the disclosures of which areincorporated herein by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates generally to the synthesis of fluorescentcompounds that are analogues of cyanine dyes. The compounds of theinvention are fluorophores that are derivatized to allow their facileattachment to another moiety. The invention also relates to improvedmethods for sequencing and genotyping nucleic acid in a single moleculeconfiguration. An exemplary method involves detection of singlemolecules of fluorescent labels released from a nucleic acid duringsynthesis of an oligonucleotide.

2. Background

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological substances as analytes in research and diagnostic mixtures.Of particular value are methods for measuring small quantities ofnucleic acids, peptides, saccharides, pharmaceuticals, metabolites,microorganisms and other materials of diagnostic value. Examples of suchmaterials include narcotics and poisons, drugs administered fortherapeutic purposes, hormones, pathogenic microorganisms and viruses,peptides, e.g., antibodies and enzymes, and nucleic acids, particularlythose implicated in disease states.

The presence of a particular analyte can often be determined by bindingmethods that exploit the high degree of specificity, which characterizesmany biochemical and biological systems. Frequently used methods arebased on, for example, antigen-antibody systems, nucleic acidhybridization techniques, and protein-ligand systems. In these methods,the existence of a complex of diagnostic value is typically indicated bythe presence or absence of an observable “label” which is attached toone or more of the interacting materials. The specific labeling methodchosen often dictates the usefulness and versatility of a particularsystem for detecting an analyte of interest. Preferred labels areinexpensive, safe, and capable of being attached efficiently to a widevariety of chemical, biochemical, and biological materials withoutsignificantly altering the important binding characteristics of thosematerials. The label should give a highly characteristic signal, andshould be rarely, and preferably never, found in nature. The labelshould be stable and detectable in aqueous systems over periods of timeranging up to months. Detection of the label is preferably rapid,sensitive, and reproducible without the need for expensive, specializedfacilities or the need for special precautions to protect personnel.Quantification of the label is preferably relatively independent ofvariables such as temperature and the composition of the mixture to beassayed.

A wide variety of labels have been developed, each with particularadvantages and disadvantages. For example, radioactive labels are quiteversatile, and can be detected at very low concentrations. However, suchlabels are expensive, hazardous, and their use requires sophisticatedequipment and trained personnel. Thus, there is wide interest innon-radioactive labels, particularly in labels that are observable byspectrophotometric, spin resonance, and luminescence techniques, andreactive materials, such as enzymes that produce such molecules.

Labels that are detectable using fluorescence spectroscopy are ofparticular interest because of the large number of such labels that areknown in the art. Moreover, as discussed below, the literature isreplete with syntheses of fluorescent labels that are derivatized toallow their attachment to other molecules, and many such fluorescentlabels are commercially available.

Fluorescent nucleic acid probes are important tools for geneticanalysis, in both genomic research and development, and in clinicalmedicine. As information from the Human Genome Project accumulates, thelevel of genetic interrogation mediated by fluorescent probes willexpand enormously. One particularly useful class of fluorescent probesincludes self-quenching probes, also known as fluorescence energytransfer probes, or FET probes. The design of different probes usingthis motif may vary in detail. In an exemplary FET probe, both afluorophore and a quencher are tethered to a nucleic acid. The probe isconfigured such that the fluorophore is proximate to the quencher andthe probe produces a signal only as a result of its hybridization to anintended target. Despite the limited availability of FET probes,techniques incorporating their use are rapidly displacing alternativemethods.

To enable the coupling of a fluorescent label with a group ofcomplementary reactivity on a carrier molecule, a reactive derivative ofthe fluorophore is prepared. For example, Reedy et al. (U.S. Pat. No.6,331,632) describe cyanine dyes that are functionalized at anendocyclic nitrogen of a heteroaryl moiety with hydrocarbon linkerterminating in a hydroxyl moiety. The hydroxyl moiety is converted tothe corresponding phosphoramidite, providing a reagent for conjugatingthe cyanine dye to a nucleic acid. Waggoner (U.S. Pat. No. 5,627,027)has prepared derivatives of cyanine and related dyes that include areactive functional group through which the dye is conjugated to anotherspecies. The compounds set forth in Ohno et al. (U.S. Pat. No.5,106,990) include cyanine dyes that have a C₁-C₅ hydrocarbyl linkerterminated with a sulfonic acid, a carboxyl or a hydroxyl group. Randallet al. (U.S. Pat. Nos. 6,197,956; 6,114,350; 6,224,644; and 6,437,141)disclose cyanine dyes with a linker arm appended to an endocyclicheteroaryl nitrogen atom. The linkers include a thiol, amine or hydroxylgroup, or a protected analogue of these residues. Additional linkerarm-cyanine dyes are disclosed by Brush et al. (U.S. Pat. Nos.5,808,044; 5,986,086). These cyanine dyes are derivatized at bothendocyclic heteroaryl nitrogen atoms with a hydrocarbyl linkerterminating in a hydroxyl moiety. One hydroxyl moiety is converted tothe corresponding phoshporamidite and the other is protected as adimethoxytrityl ether.

Cyanine dyes are particularly popular fluorophores and are widely usedin many biological applications due to their high quantum yield and highmolar absorbtivity. Cyanine dyes are, however, susceptible tophotobleaching during prolonged excitation. Moreover, due the rigidplanar structure of these compounds, they have a tendency to stack andself-quench. Thus, provision of cyanine dyes having an enhancedbrightness and decreased tendency to stack, thereby mitigating theeffects of photobleaching and stacking is an important object.Furthermore, cyanine dyes that are hydrophilic are less attracted toother species such as proteins and surfaces, which reduces adventitiousbinding of the fluorophore and enhances the precision and accuracy ofassays and other analyses utilizing cyanine fluorophores. The presentinvention meets these objects and other needs.

SUMMARY OF THE INVENTION

The present invention provides a class of cyanine-based fluorophoresmodified to improve their fluorescent and other physicochemicalproperties. Thus, it is a general object of the invention to providecyanine dyes that are hydrophilic, are resistant to photobleaching, ormaintain a high level of brightness despite photobleaching, and have alower tendency to stack or otherwise aggregate than current cyaninefluorophores.

Exemplary dyes of the invention find particular use in DNA sequencingmodalities, particularly single molecule sequencing modalities. Previousdyes used in such applications have had less than ideal properties. Forexample, certain dyes give suboptimal performance, because, as wasdiscovered, the dyes are insufficiently hydrophilic, insufficientlybright, do not emit steadily (i.e., blink), undergo photobleaching uponprolonged irradiation or they aggregate. These deficiencies can causemisreads in DNA sequencing analyses, providing inaccurate results. Invarious embodiments, the present invention provides a solution to one ormore of these factors contributing to suboptimal dye performance. Invarious embodiments, the hydrophilicity of the dyes is enhanced by theaddition of to the cyanine core or a side group attached to the cyaninecore of a water-soluble polymer, sulfonic acid, or carboxylic acidmoieties or groups containing sulfonic acid or carboxylic acid moieties.Moreover, it was discovered that substitution of a cyanine dye withcharged, hydrophilic moieties protects the cyanine chromophore from thedye's microenvironment and reduces blinking, aggregation andphotobleaching. Thus, in various embodiments, the dyes are brighter,more photostable and their emission is more constant. Furthermore, forDNA sequencing, particularly single molecule sequencing, resolution ofthe absorbance of the dye emissions is important to sensitivity andaccuracy of the measurements underlying the sequence determination.Accordingly, in various embodiments, the present invention provides dyeswith emissions tuned to achieve useful levels of resolution in theemission peaks of the dyes when they are used in combinations of 2, 3, 4or more different dyes attached to nucleic acids. Thus, in variousembodiments, the present invention provides a solution to the problem.In exemplary embodiments, the dyes of the invention provide at least a2%, at least a 5%, at least a 7% or at least a 10% improvement inreadlength in a single molecule DNA sequencing protocol when comparedwith dyes that are not functionalized as are the dyes of the invention.

In exemplary embodiments, the dyes of the invention are utilized in DNAsequencing in real time using a single polymerase enzyme attached to thebottom of the small nano-meter size hole called zero-mode waveguide(ZMW). Fluorescent signals of 4 different colors that correspond to 4different DNA bases: A, G, C, T are detected. Since the most robustmethodologies read through as many bases on a template oligonucleotideas possible, it is desirable to utilize dyes that do not limit thereadlength or the accuracy of the measurements. The water-soluble,cyanine dyes of the invention are of use in such measurements and insome embodiments increase the accuracy of the measurements by at least2%, at least 5%, at least 7% or at least 10% in a single molecule DNAsequencing protocol when compared with dyes that are not functionalizedas are the dyes of the invention.

In an exemplary embodiment, the dyes of the invention include a rigidlinker arm with a peptide backbone. The peptide provides a versatilelinker arm, the structure and position of which is readily alterable,thereby allowing the conjugation of the label through a variety ofpositions on the cyanine nucleus to a carrier molecule. Exemplaryspecies to which the amino acid or peptide linked fluorophores are boundinclude nucleic acids and polyvalent moieties (e.g., a scaffold). Choiceof the amino acid or peptide constituent of the linker influences theproperties of the conjugate. For example, selection of an amino acid orpeptide linker was found to allow the strength and time course of theinteraction between the fluorescent nucleic acid analogue to be varied.In exemplary embodiments, the amino acid or peptide moiety enhances theinteraction between the compound of the invention and a protein such asa DNA polymerase, lowering the K_(off) of the sequencing reaction.

The versatility of the labels set forth herein provides a markedadvantage over currently utilized cyanine labels, probes assembled usingthose labels and methods relying upon such labels and probes. Moreover,the present invention provides a class of chemically versatile labels inwhich the fluorophore can be engineered to have a desired lightexcitation and emission profile.

In various embodiments, the present invention provides a class ofconjugates that include fluorophores bound to an amino acid or peptide.In exemplary embodiments, the amino acid or peptide serves as a linkerand is itself bound to another species.

In an exemplary embodiment, the present invention provides a fluorescentdye having the formula:

{R¹-(L¹)_(a)-(AA)_(n)}_(y)-(L²)_(b)-X  (I)

wherein R¹ is a fluorescent dye moiety. AA is an amino acid. The index nis selected from the integers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12,and when n is two or greater, each n amino acid is independentlyselected. X is a member selected from a polyvalent moiety, and a moietyincluding the structure:

wherein Y is a nucleobase; and u is selected from the integers 1, 2, 3,4, 5, 6, 7 and 8. The index y is selected from the integers 1, 2, 3, 4,5, 6, 7 and 8, such that when y is 2 or greater, X is a polyvalentmoiety. L¹ and L² are independently selected from bonds, adaptors andsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl moieties. The index a is 0 or 1, and b is an integerselected from 0, 1, 2, 3, 4, 5, 6, 7 and 8.

In various embodiments, the invention provides compounds in which thefluorescent dye moiety has the formula:

A and B independently selected monocyclic, bicyclic or polycyclic arylor heteroaryl moieties. When A and/or B is a bicyclic polycyclic moiety,two or more of the rings are optionally fused. Exemplary polycyclicmoieties include indole and benzoindole. Q is a substituted orunsubstituted methine moiety (e.g., —(CH═C(R))_(c)—CH═), in which c isan integer selected from 1, 2, 3, 4, or 5 and R is an “alkyl groupsubstituent” as defined herein. When two or more R groups are present,they are optionally joined to form a ring. Each R^(w), R^(x), R^(y) andR^(z) is independently selected from those substituents set forth in theDefinitions section herein as “alkyl group substituents” and “aryl groupsubstituents.” The indices w and z are independently selected from theintegers from 0 to 6. In an exemplary embodiment, at least one of R^(w),R^(x), R^(y) and R^(z) is C(O)NR^(o)(CH₂)_(h)G in which G is a memberselected from SO₃H and CO₂H, R^(o) is H or substituted or unsubstitutedalkyl or heteroalkyl and the index h is an integer from 1 to 20. Inexemplary embodiments, at least 1, 2, 3, 4, 5, or 6 of R^(x), R^(y),R^(w) and R^(z) are alkylsulfonic acid or heteroalkylsulfonic acid andat least one of these moieties is alkylcarboxylic acid orheteroalkylcarboxylic acid. In exemplary embodiments, at least one ofR^(w), R^(x), R^(y) and R^(z) includes a water-soluble polymer (e.g.,poly(ethylene glycol)) component.

In various embodiments, at least one of R^(w), R^(x), R^(y) and R^(z) isfunctionalized with an additional dye moiety bonded to the cyanine dyecore shown above. In an exemplary embodiment, the additional dye moietyis bonded to the dye core through a linker, a polyvalent scaffold, or alinker-polyvalent scaffold conjugate.

In various embodiments, the invention provides a composition, comprisingan enzyme, and a substrate for the enzyme, the substrate comprising acomponent reacted upon by the enzyme, a fluorescent label component andan amino acid or peptide linker component conjugating these twocomponents. The linker component interacts with the enzyme to increasethe affinity of the flurophore-linker-enzyme reactive component with theenzyme, reducing the K_(m) of the reaction between the enzyme and theenzyme-reactive component relative to that of an analogous reaction inwhich the conjugate does not include the linker component. Exemplaryinteraction modalities by which the linker increases the affinity of theconjugate for the enzyme include, without limitation, electrostatic,hydrophobic and steric interactions. In various embodiments, the K_(m)is reduced at least 10%, at least 20%, at least 30%, at least 40% or atleast 50% relative to the K_(m) of the reaction with an analogousconjugate without the linker component.

In a further aspect, the invention provides a method of monitoring anenzyme reaction. The method generally comprises providing a reactionmixture comprising the enzyme and at least a first reactant composition.An exemplary reactant composition comprises a compound having acomponent that reacts with the enzyme, a fluorescent label component,and an adaptor or linker-adaptor component joining the reactantcomponent to the label component. The reaction mixture is thenilluminated to excite the fluorescent label component, and a fluorescentsignal from the reaction mixture characteristic of the enzyme reactionis detected.

The invention also provides methods of monitoring nucleic acid synthesisreactions. The methods comprise contacting a polymerase/template/primercomplex with a fluorescently labeled nucleotide or nucleotide analoghaving a nucleotide or nucleotide analog component, a fluorescent labelcomponent, and an adaptor or linker-adaptor component joining dienucleotide or nucleotide analog component to the label component. Acharacteristic signal from the fluorescent dye is then detected that isindicative of incorporation of the nucleotide or nucleotide analog intoa primer extension reaction.

In various embodiments, the present invention provides methods of usingthe compounds described herein for performing nucleic acid analyses, andparticularly nucleic acid sequence analyses. In various embodiments, thecompounds of the invention are used in single molecule nucleic acidsequencing. Exemplary methods of the invention comprise using a templatenucleic acid complexed with a polymerase enzyme in a template dependentpolymerization reaction to produce a nascent nucleic acid strand,contacting the polymerase and template nucleic acid with a compound ofthe invention, and detecting whether or not the compound or asubstructure thereof (e.g., a monophosphate nucleic acid) wasincorporated into the nascent strand during the polymerization reaction,and identifying a base in the template strand based upon incorporationof the compound. Preferably, the foregoing process is carried out so asto permit observation of individual nucleotide incorporation reactions,through the use of, for example, an optical confinement, that allowsobservation of an individual polymerase enzyme, or through the use of aheterogeneous assay system, where fluorophores released fromincorporated analogs are detected.

The compounds and compositions of the invention are of use in singlemolecule or single molecule real time (SMRT) DNA sequencing assays. Ofparticular note in this context is the ability provided by the inventionto design fluorophores with selected absorbance and emission propertiesincluding wavelength and intensity. The compounds of the inventionprovide for very versatile assay design. For example, according to thepresent invention a series of fluorophores of use in an assay arereadily designed to have selected absorbance and emission wavelengthsand emission intensities, allowing multiple fluorophores to be utilizedand distinguished in an assay. In exemplary embodiments, use ofcompounds of the invention in a multifluorophore assay, e.g., singlemolecule DNA sequencing, enhances assay performance by at least about10%, at least about 20% or at least about 30% over a similar assay usingcurrently available fluorophores.

Other aspects, embodiments and objects of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a), FIG. 1( b) and FIG. 1( c) show structures of exemplaryprecursors of the dye components of the conjugates of the invention.Once incorporated into a conjugate of the invention, the conjugated dyescan be further conjugated to one or more additional species, e.g., apolyvalent scaffold (e.g., into a FRET pair), conjugated to a nucleicacid or to a linker.

FIG. 2( a), FIG. 2( b) and FIG. 2( c) show structures of exemplaryprecursors of the dye components of the conjugates of the invention.Once incorporated into a conjugate of the invention, the conjugated dyescan be further conjugated to one or more additional species, e.g., apolyvalent scaffold (e.g., into a FRET pair), conjugated to a nucleicacid or to a linker.

FIG. 3( a) and FIG. 3( b) show structures of exemplary precursors of thedye components of the conjugates of the invention. Once incorporatedinto a conjugate of the invention, the conjugated dyes can be furtherconjugated to one or more additional species, e.g., a polyvalentscaffold (e.g., into a FRET pair), conjugated to a nucleic acid or to alinker.

FIG. 4( a) is a generic structure of exemplary precursors of the dyecomponents of the conjugates of the invention and of substituents onthese precursors. Once incorporated into a conjugate of the invention,the conjugated dyes can be further conjugated to one or more additionalspecies, e.g., a polyvalent scaffold (e.g., into a FRET pair),conjugated to a nucleic acid or to a linker. FIG. 4( b) is a tabulationof exemplary dye component precursors according to the generic structureof FIG. 4( a).

FIG. 5( a) is a generic structure of exemplary precursors of the dyecomponents of the conjugates of the invention and of substituents onthese precursors. Once incorporated into a conjugate of the invention,the conjugated dyes can be further conjugated to one or more additionalspecies, e.g., a polyvalent scaffold (e.g., into a FRET pair),conjugated to a nucleic acid or to a linker. FIG. 5( b) is a tabulationof exemplary dye component precursors according to the generic structureof FIG. 5( a).

FIG. 6( a) is a generic structure of exemplary precursors of the dyecomponents of the conjugates of the invention and of substituents onthese precursors. Once incorporated into a conjugate of the invention,the conjugated dyes can be further conjugated to one or more additionalspecies, e.g., a polyvalent scaffold (e.g., into a FRET pair),conjugated to a nucleic acid or to a linker. FIG. 6( b) is a tabulationof exemplary dye component precursors according to the generic structureof FIG. 6( a).

FIG. 7( a) is a generic structure of exemplary precursors of the dyecomponents of the conjugates of the invention and of substituents onthese precursors. Once incorporated into a conjugate of the invention,the conjugated dyes can be further conjugated to one or more additionalspecies, e.g., a polyvalent scaffold (e.g., into a FRET pair),conjugated to a nucleic acid or to a linker. FIG. 7( b) is a tabulationof exemplary dye component precursors according to the generic structureof FIG. 7( a).

FIG. 8( a) is a generic structure of exemplary precursors of the dyecomponents of the conjugates of the invention and of substituents onthese precursors. Once incorporated into a conjugate of the invention,the conjugated dyes can be further conjugated to one or more additionalspecies, e.g., a polyvalent scaffold (e.g., into a FRET pair),conjugated to a nucleic acid or to a linker. FIG. 8( b) is a tabulationof exemplary dye component precursors according to the generic structureof FIG. 8( a).

FIG. 9( a) is a generic structure of exemplary precursors of the dyecomponents of the conjugates of the invention and of substituents onthese precursors. Once incorporated into a conjugate of the invention,the conjugated dyes can be further conjugated to one or more additionalspecies, e.g., a polyvalent scaffold (e.g., into a FRET pair),conjugated to a nucleic acid or to a linker. FIG. 9( b) is a tabulationof exemplary dye component precursors according to the generic structureof FIG. 9( a).

FIG. 10( a) is a generic structure of exemplary precursors of the dyecomponents of the conjugates of the invention and of substituents onthese precursors. Once incorporated into a conjugate of the invention,the conjugated dyes can be further conjugated to one or more additionalspecies, e.g., a polyvalent scaffold (e.g., into a FRET pair),conjugated to a nucleic acid or to a linker. FIG. 10( b) is a tabulationof exemplary dye component precursors according to the generic structureof FIG. 10( a).

FIG. 11( a) is a generic structure of exemplary precursors of the dyecomponents of the conjugates of the invention and of substituents onthese precursors. Once incorporated into a conjugate of the invention,the conjugated dyes can be further conjugated to one or more additionalspecies, e.g., a polyvalent scaffold (e.g., into a FRET pair),conjugated to a nucleic acid or to a linker. FIGS. 11( b) and 11(c) is atabulation of exemplary dye component precursors according to thegeneric structure of FIG. 11( a).

FIGS. 12( a) and 12(b) display structures of exemplary nucleic acid(polyphosphate) conjugates of the invention.

FIGS. 13( a) and 13(b) display structures of exemplary monovalent andpolyvalent dye nucleic acid (polyphosphate) conjugates of the invention.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

“FET”, as used herein, refers to “Fluorescence Energy Transfer.”

“FRET”, as used herein, refers to “Fluorescence Resonance EnergyTransfer.” These terms are used herein to refer to both radiative andnon-radiative energy transfer processes. For example, processes in whicha photon is emitted and those involving long-range electron transfer areincluded within these terms. Throughout this specification, both ofthese phenomena are subsumed under the general term “donor-acceptorenergy transfer.”

Any of the dyes set forth herein can be a component of an FET or FRETpair as either the donor or acceptor. Conjugating a compound of theinvention and a donor or acceptor fluorophore through reactivefunctional groups on the conjugation partners and an appropriate linker,adaptor, carrier molecule or a combination thereof is well within theabilities of those of skill in the art.

The symbol “R”, as used herein, refers to moiety which is a memberselected from the moieties defined in the following section, e.g.,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, etc. as well as those groups set forth assubstituents of these moieties.

As used herein, the abbreviations for the genetically encodedL-enantiomeric amino acids used in the linker constructs in thecompounds of the invention are conventional and are as follows:

One-Letter Common Amino Acid Symbol Abbreviation Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

DEFINITIONS

Where chemical moieties are specified by their conventional chemicalformulae, written from left to right, they optionally equally encompassthe moiety which would result from writing the structure from right toleft, e.g., —CH₂O— is intended to also recite —OCH₂—; —NHS(O)₂— is alsointended to optionally represent. —S(O)₂HN—, etc. Moreover, wherecompounds can be represented as free acids or free bases or saltsthereof, the representation of a particular form, e.g., carboxylic orsulfonic acid, also discloses the other form, e.g., the deprotonatedsalt form, e.g., the carboxylate or sulfonate salt. Appropriatecounterions for salts are well-known in the art, and the choice of aparticular counterion for a salt of the invention is well within theabilities of those of skill in the art. Similarly, where the salt isdisclosed, this structure also discloses the compound in a free acid orfree base form. Methods of making salts and free acids and free basesare well-known in the art.

“Amino Acid,” as used herein refers to the genus encompassinghydrophilic amino acids, acidic amino acids, basic amino acids, polaramino acids, hydrophobic amino acids, aromatic amino acids, non-polaramino acids and aliphatic amino acids, including the genus and thespecies therein. The peptide linkers of the invention are formed fromsuch amino acids. Amino acids also encompass amino-carboxylic acidspecies other than α-amino acids, e.g., aminobutyric acid (aba),aminohexanoic acid (aha), aminomethylbenzoic acid (amb) etc.

“Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilic amino acids include Thr (T),Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg I.

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg I and Lys (K).

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q), Ser (S) and Thr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:125-142. Exemplary hydrophobic amino acids include Ile (I), Phe (F),Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), Pro (P),and proline analogues.

“Aromatic Amino Acid” refers to a hydrophobic amino acid with a sidechain having at least one aromatic or heteroaromatic ring. The aromaticor heteroaromatic ring may contain one or more substituents such as —OH,—SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂, —NHR, —NRR, —C (O)R,—C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR and the like where each Ris independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl, (C₁-C₆)alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted(C₁-C₆) alkynyl, (C₁-C₂₁)) aryl, substituted (C₅-C₂₀) aryl, (C₆-C₂₆)alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl,substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl orsubstituted 6-26 membered alkheteroaryl. Genetically encoded aromaticamino acids include Phe (F), Tyr (Y) and Trp (W).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded apolar amino acids include Leu (L), Val (V), Ile(I), Met (M), Gly (G) and Ala (A).

“Aliphatic Amino Acid” refers to a hydrophobic amino acid having analiphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala (A), Val (V), Leu (L) and Ile (I).

Peptide linkers in the compounds of the invention are formed from aminoacids linked by one or more peptide bond. The linkers are formed fromoligomers of the same amino acid or different amino acids.

An “Adaptor” is a moiety that is at least bivalent. Exemplary adaptorsare bound to a nucleic acid and a fluorescent dye, either directly orthrough a linker. The adaptor can also be bound to a second fluorescentdye, to a polyvalent scaffold or to a second nucleic acid. When theadaptor is bound to a second dye, either directly or through apolyvalent scaffold, the resulting conjugate is optionally a FRET pair.The adaptor is preferably bound to the phosphorus atom of a phosphate,phosphate ester or polyphosphate moiety of a nucleic acid. In exemplaryembodiments, the adaptor is bound through an amide moiety to the dye orto the linker of the linker-dye cassette. The amide moiety is formedbetween an amine on the adaptor and a carboxyl group on the dye or thelinker precursor.

“Cyanine,” as used herein, refers to aryl and heteroaryl polymethinedyes such as those based upon the cyanine, merocyanine, styryl andoxonol ring.

As used herein, “nucleic acid” means any natural or non-naturalnucleoside, or nucleotide and oligomers and polymers thereof, e.g., DNA,RNA, single-stranded, double-stranded, triple-stranded or more highlyaggregated hybridization motifs, and any chemical modifications thereof.Modifications include, but are not limited to, conjugation into acompound of the invention. Further modifications include those providingthe nucleic acid with a group that incorporates additional charge,polarizability, hydrogen bonding, electrostatic interaction,fluxionality or functionality to the nucleic acid. Exemplarymodifications include the attachment to the nucleic acid, at anyposition, of one or more hydrophobic or hydrophilic moieties, minorgroove binders, intercalating agents, quenchers, chelating agents, metalchelates, solid supports, and other groups that are usefully attached tonucleic acids. Exemplary nucleic acids of the invention include one ormore dye moiety of the invention bound thereto.

Exemplary modified nucleic acids include, but are not limited to,peptide nucleic acids (PNAs), those with phosphodiester groupmodifications (e.g., replacement of O⁻ with OR, NR, or SR), 2′-, 3′- and5′-position sugar modifications, modifications to the nucleobase moiety,e.g., 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, i.e., substitution of P(O)O₃ with another moiety,methylations, unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine and the like. Nucleic acids can alsoinclude non-natural bases, e.g., nitroindole. Non-natural nucleobasesinclude bases that are modified with a compound of the invention or alinker-compound of the invention construct, a minor groove binder, anintercalating agent, a hybridization enhancer, a chelating agent, ametal chelate, a quencher, a fluorophore, a fluorogenic compound, etc.Modifications within the scope of “nucleic acid” also include 3′ and 5′modifications with one or more of the species described above.

The nucleic acid can comprise DNA, RNA or chimeric mixtures orderivatives or modified versions thereof. Both the probe and targetnucleic acid can be present as a single strand, duplex, triplex, etc.Moreover, as discussed above, the nucleic acid can be modified at thenucleobase moiety, sugar moiety, or phosphate backbone with other groupssuch as radioactive labels, minor groove binders, intercalating agents,donor and/or acceptor moieties and the like.

In addition to the naturally occurring “nucleobases,” adenine, cytosine,guanine and thymine, nucleic acid components of the compounds of theinvention optionally include modified bases. These components can alsoinclude modified sugars. For example, the nucleic acid can comprise atleast one modified base moiety which is selected from the groupincluding, but not limited to, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,nitroindole, and 2,6-diaminopurine. The dye of the invention or anotherprobe component can be attached to the modified base.

In another embodiment, the nucleic acid comprises at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose. The dye or anotherprobe component can be attached to the modified sugar moiety.

In yet another embodiment, the nucleic acid comprises at least onemodified phosphate backbone selected from the group including, but notlimited to, a peptide nucleic acid hybrid, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof. The dye or another probe component can beattached to the modified phosphate backbone.

“Nucleic acid” also includes a component of a conjugte with one or moremodified phosphate bridges (e.g., P(O)O₃) by conjugating a linker-dyeconjugate of the invention to the nucleic acid, e.g., replacing orderivatizing an oxygen of the bridge) with a compound of the inventionor a species that includes a compound of the invention attached to anadaptor. For example, “nucleic acid” also refers to species in which,rather than the P(O)(O⁻)O₂ moiety of a naturally occurring nucleic acid,includes the moiety ROP(O)(O—)O, in which R is a dye-linker conjugate ofthe invention, an adaptor, a linker-adaptor cassette or a fluorescentdye-linker-adaptor cassette. An exemplary linker is an amino acid orpeptide linker of the invention. In various embodiments, one oxygen ofthis structure is bound to the phosphorus atom of a P(O)(O⁻)O₂, suchthat the nucleic acid includes two or more phosphate moieties bound toeach other.

Further exemplary nucleic acids of the invention include a nucleotidehaving a polyphosphate moiety, e.g., pyrophosphate or a higherhomologue, such as the 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer and thelike. Exemplary nucleic acids include such a polyphosphate moiety bondedto the 5′-oxygen of a nucleoside. In addition to the attachedpolyphosphate moiety can include a modified phosphate bridge, such asthose exemplified herein. In an exemplary embodiment, the modifiedphosphate bridge is modified with an adaptor, a linker dye conjugate, alinker-adaptor cassette or a fluorescent dye-linker-adaptor cassette. Inan exemplary embodiment, the linker is an amino acid or peptide linkersuch as those set forth herein. Examples of some nucleic acids findinguse in the present invention are set forth in Published U.S. PatentApplication Nos. 2003/0124576 and 2007/0072196 as well as U.S. Pat. Nos.7,223,541 and 7,052,839, the full disclosures of which are incorporatedherein by reference for all purposes.

Furthermore, “nucleic acid” includes those species in which one or moreinternucleotide bridge does not include phosphorus: the bridge beingoptionally modified with a compound of the invention or a linker-dyeconstruct of the invention. An exemplary bridge includes a substitutedor unsubstituted alkyl or substituted or unsubstituted heteroalkylmoiety in which a carbon atom is the locus for the interconnection oftwo nucleoside sugar residues (or linker moieties attached thereto) anda linker-dye construct of the invention. The discussion above is notlimited to moieties that include a carbon atom as the point ofattachment; the locus can also be another appropriate linking atom, suchas nitrogen or another atom.

Phosphodiester linked nucleic acids of the invention can be synthesizedby standard methods known in the art, e.g. by use of an automated DNAsynthesizer using commercially available amidite chemistries (Ozaki etal., Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al.,Nucleic Acids Research, 18: 5419-5423 (1990); Beaucage et al.,Tetrahedron, 48: 2223-2311 (1992); Molko et al., U.S. Pat. No.4,980,460; Koster et al., U.S. Pat. No. 4,725,677; Caruthers et al.,U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679). Nucleic acidsbearing modified phosphodiester linking groups can be synthesized bymethods known in the art. For example, phosphorothioate nucleic acidsmay be synthesized by the method of Stein et al. (Nucl. Acids Res.16:3209 (1988)), methylphosphonate nucleic acids can be prepared by useof controlled pore glass polymer supports (Sarin et al., Proc. Natl.Acad. Sci. U.S.A. 85:7448-7451 (1988)). Other methods of synthesizingboth phosphodiester- and modified phosphodiester-linked nucleic acidswill be apparent to those of skill in the art.

As used herein, “quenching group” refers to any fluorescence-modifyinggroup of the invention that can attenuate, at least partly, the energy(e.g., light) emitted by a fluorescent dye. This attenuation is referredto herein as “quenching”. Hence, irradiation of the fluorescent dye inthe presence of the quenching group leads to an emission signal from thefluorescent dye that is less intense than expected, or even completelyabsent. Quenching typically occurs through energy transfer between thefluorescent dye and the quenching group.

“Carrier molecule,” as used herein refers to any molecule to which acompound of the invention, or a conjugate incorporating a compound ofthe invention, is attached. Representative carrier molecules include anucleic acid, protein (e.g., enzyme, antibody), glycoprotein, peptide,saccharide (e.g., mono-, oligo-, and poly-saccharides), hormone,receptor, antigen, substrate, metabolite, transition state analog,cofactor, inhibitor, drug, dye, nutrient, growth factor, etc., withoutlimitation. “Carrier molecule” also refers to species that might not beconsidered to fall within the classical definition of “a molecule,”e.g., solid support (e.g., synthesis support, chromatographic support,membrane), virus and microorganism. An exemplary carrier molecule of usein the present invention is a polyphosphate nucleic acid. Exemplaryconjugates between a fluorescent dye and a polyphosphate nucleic acidare conjugated by covalent binding of the dye to the linker and hence tothe nucleic acid, or covalent binding of the dye to a linker and thelinker to the adaptor—the adaptor is conjugated to the nucleic acid.Alternatively, the dye is bound to a linker, which is bound to anadaptor, which is bound to the nucleic acid. In an exemplary embodiment,the adaptor is bound to the polyphosphate moiety through aphosphodiester bond. In an exemplary embodiment, the adaptor (or linker)is attached to the dye through a bond formed with an activatedderivative of a carboxyl moiety on the dye. In various embodiments, thebond is an amide bond.

“Activated derivatives of carboxyl moieties,” and equivalent species,refers to moiety on a precursor component of a conjugate of theinvention (e.g., dye, adaptor, linker, polyvalent moiety) having anoxygen-containing, or other, leaving group, e.g., an active ester, acylhalide, acyl imidazolide, etc.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturated alkylradicals include, but are not limited to, groups such as methyl,methylene, ethyl, ethylene, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl, and the like. An unsaturated alkyl group is one having one ormore double bonds or triple bonds. Examples of unsaturated alkyl groupsinclude, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, includes “alkylene”and, optionally, those derivatives of alkyl defined in more detailbelow, such as “heteroalkyl.”

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, S, P and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Also included aredi- and multi-valent species such as “cycloalkylene.” Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to, speciessuch as trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings), which are fused togetheror linked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from one to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. A heteroaryl group canbe attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Also included are di- and multi-valentlinker species, such as “arylene.” Substituents for each of the abovenoted aryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxyl)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Exemplary substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, SO₃R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR′″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. Accordingly,from the above discussion of substituents, one of skill in the art willunderstand that the terms “substituted alkyl” and “heteroalkyl” aremeant to include groups that have carbon atoms bound to groups otherthan hydrogen atoms, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl(e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The substituents set forth in the paragraph above are referred to hereinas “alkyl group substituents.”

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, SO₃R′, —S(O)₂NR′R″, —NRSO₂R′, —CNand —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R′are preferably independently selected from hydrogen, (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl and heteroaryl, (unsubstitutedaryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

The substituents set forth in the two paragraphs above are referred toherein as “aryl group substituents.”

“Analyte”, “target”, “substance to be assayed”, and “target species,” asutilized herein refer to the species of interest in an assay mixture.The terms refer to a substance, which is detected qualitatively orquantitatively using a material, process or device of the presentinvention. Examples of such substances include cells and portionsthereof, enzymes, antibodies, antibody fragments and other biomolecules,e.g., antigens, polypeptides, glycoproteins, polysaccharides, complexglycolipids, nucleic acids, effector molecules, receptor molecules,enzymes, inhibitors and the like and drugs, pesticides, herbicides,agents of war and other bioactive agents.

More illustratively, such substances include, but are not limited to,tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), CA125, CA 19-9 and the like; various proteins, glycoproteins and complexglycolipids such as β₂-microglobulin (β₂ m), ferritin and the like;various hormones such as estradiol (E₂), estriol (E₃), human chorionicgonadotropin (hCG), luteinizing hormone (LH), human placental lactogen(hPL) and the like; various virus-related antigens and virus-relatedantibody molecules such as HBs antigen, anti-HBs antibody, HBc antigen,anti-HBc antibody, anti-HCV antibody, anti-HIV antibody and the like;various allergens and their corresponding IgE antibody molecules;narcotic drugs and medical drugs and metabolic products thereof; andnucleic acids having virus- and tumor-related polynucleotide sequences.

The term, “assay mixture,” refers to a mixture that includes the analyteand other components. The other components are, for example, diluents,buffers, detergents, and contaminating species, debris and the like thatare found mixed with the target. Illustrative examples include urine,sera, blood plasma, total blood, saliva, tear fluid, cerebrospinalfluid, secretory fluids from nipples and the like. Also included aresolid, gel or sol substances such as mucus, body tissues, cells and thelike suspended or dissolved in liquid materials such as buffers,extractants, solvents and the like.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesof be composed of a single amino acid, e.g., poly(lysine). An exemplarypolysaccharide is poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol), e.g., m-PEG. Poly(ethylene imine) is an exemplarypolyamine, and poly(acrylic) acid is a representative poly(carboxylicacid).

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH).sub.m in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as polypropylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

The term PEG or poly(ethylene glycol) is intended to be inclusive andnot exclusive. The term PEG includes poly(ethylene glycol) in any of itsforms, including alkoxy PEG, difunctional PEG, multiarmed PEG, forkedPEG, branched PEG, pendent PEG (i.e., PEG or related polymers having oneor more functional groups pendent to the polymer backbone), or PEG withdegradable linkages therein.

The PEG backbone can be linear or branched. Branched polymer backbonesare generally known in the art. Typically, a branched polymer has acentral branch core moiety and a plurality of linear polymer chainslinked to the central branch core. PEG is commonly used in branchedforms that can be prepared by addition of ethylene oxide to variouspolyols, such as glycerol, pentaerythritol and sorbitol. The centralbranch moiety can also be derived from several amino acids, such aslysine. The branched poly(ethylene glycol) can be represented in generalform as R(-PEG-OH)_(m) in which R represents the core moiety, such asglycerol or pentaerythritol, and m represents the number of arms.Multi-armed PEG molecules, such as those described in U.S. Pat. No.5,932,462, which is incorporated by reference herein in its entirety,can also be used as the polymer backbone.

An “Adaptor” is a moiety that is at least bivalent and which is bound toa linker bound to a dye or it is bound directly to the dye. The adaptoralso forms a bond with a second dye, polyvalent scaffold or to a nucleicacid. When the adaptor is bound to another dye, either directly orthrough a polyvalent scaffold, the resulting conjugate is optionally aFRET pair. When the adaptor is bound to a nucleic acid, it is preferablybound to the phosphorus atom of a phosphate, phosphate ester orpolyphosphate moiety. In exemplary embodiments, the adaptor is boundthrough an amide moiety to the dye. The amide moiety is formed betweenan amine on the adaptor and a carboxyl group on the dye.

“Readlength” is the number of bases the DNA polymerase enzyme at thebottom of the ZMW goes through during sequencing. A longer readlength isdesirable. Readlength depends, inter alia, on how fast the enzyme canincorporate fluorescent nucleotides of different colors (monitored thisby observing pulse widths and interpulse distances). Readlength alsodepends on how long the enzyme can incorporate analog without beingphotodamaged (damaged via undesired interactions with fluorescentnucleotides excited by light).

“Accuracy” is how precise a nucleotide with a base of a particular typecan be identified as the polymerase enzyme goes through incorporation offluorescent nucleotides. The base is identified by a pulse of a selectedwavelength upon incorporation of the nucleotide incorporating that base.Robust applications include precise base calling. Accuracy can bediminished by one or more of extra pulses, missing pulses and miscalledpulses.

“Extra pulses”—when a pulse is called and there is no nucleotideincorporation event. Extra pulses may be caused by branching (whenenzyme samples the fluorescent analog but does not incorporate), sticks(non-specific interactions of fluorescent nucleotides with enzymeoutside of incorporating site and surface of ZMW), photophysicalblinking (photophysically unstable behavior of fluorescent nucleotidesduring incorporation resulting in splitting of fluorescent signal).

“Missing pulses”—when a pulse is not called when there is in fact anucletided incorporation event. Missing pulses may be caused byinsufficient brightness of fluorescent nucleotides, low purity offluorescent nucleotides, or polymerase going too fast to detect allpulses.

“Miscalled pulses”—when pulse of different kind is called instead ofcorrect one. Miscalls may be caused by insufficient spectral separationbetween fluorescent nucleotides of different colors, photophysicalinstability of our fluorescent nucleotides, low intensity or highbackground of fluorescent nucleotide signal.

INTRODUCTION

Residing in the field of fluorescent labels, the present inventionprovides benefits of particular note. Fluorescent labels have theadvantage of requiring few precautions in handling, and being amenableto high-throughput visualization techniques (optical analysis includingdigitization of the image for analysis in an integrated systemcomprising a computer). Exemplary labels exhibit one or more of thefollowing characteristics: high sensitivity, high stability, lowbackground, low environmental sensitivity and high specificity inlabeling.

Amongst the advantages provided by the present invention are includedadvances in the field of monitoring enzymatic reactions. In exemplaryembodiments, the linker component of compounds of the inventionincreases the affinity of a conjugate of the invention, which is asubstrate for an enzyme, for this enzyme, reducing the K_(m) of thereaction. In various embodiments, the Km of the reaction is reduced byat least 10%, at least 20%, at least 30%, at least 40% or at least 50%relative to the K_(m) of the reaction with an analogous conjugatewithout the amino acid or peptide linker component.

The compounds, probes and methods discussed in the following sectionsare generally representative of the compositions of the invention andthe methods in which such compositions can be used. The followingdiscussion is intended as illustrative of selected aspects andembodiments of the present invention and it should not be interpreted aslimiting the scope of the present invention.

The Embodiments Compositions

In an exemplary embodiment, the present invention provides a fluorescentdye having the formula:

{R¹-(L¹)_(a)-(AA)_(n)}_(y)-(L²)_(b)-X  (I)

wherein R¹ is a fluorescent dye moiety. AA is an amino acid. The index nis selected from the integers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12,and when n is two or greater, each n amino acid is independentlyselected and the (AA)_(n) component is a peptide. X is a member selectedfrom a polyvalent moiety, and a moiety including the structure:

wherein Y is a nucleobase; and u is selected from the integers 1, 2, 3,4, 5, 6, 7 and 8. The index y is selected from the integers 1, 2, 3, 4,5, 6, 7 and 8, such that when y is 2 or greater, X is a polyvalentmoiety. L¹ and L² are independently selected from bonds, adaptors andsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl moieties. The index a is 0 or 1, and b is an integerselected from 0, 1, 2, 3, 4, 5, 6, 7 and 8.

In exemplary embodiments, the compound of the invention has the formula:

R¹-(L¹)_(a)-(AA)_(n)-(L²)_(b)-X  (III)

in which the radicals and indices are as discussed herein.

In various embodiments, L¹ and/or L² is, or includes an adaptorcomponent. Exemplary adaptors of use in the compounds of the inventioninclude those selected from an alkyl amine or a nitrogen-containingheterocylic moiety, e.g., piperidine. Exemplary species include anaminoalkyl (e.g. C₁-C₁₀ aminoalkyl, e.g., C₆ aminoalkyl) linker,—NH(CH₂)_(g)C(O)NH(CH₂)_(h)—, in which g and h are independentlyselected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher.Such adaptors include, without limitation:

in which q is the integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In variousembodiments, the nitrogen of the amine moiety is bound to a fluorescentdye, a linker or a linker bound to a fluorescent dye. The other openvalence is bound to the group X shown in Formula I.

In various embodiments, the adaptor has a formula selected from:

in which q is as described above. In various embodiments, the nitrogenof the amine moiety is bound to a fluorescent dye, a linker or a linkerbound to a fluorescent dye. The oxygen atom is bound to the group Xshown in Formula I.

In exemplary embodiments, the adaptor has a formula selected from:

in which q is as described above. In various embodiments, the nitrogenof the amine moiety is bound to a fluorescent dye, a linker or a linkerbound to a fluorescent dye. The oxygen atom is bound to the group Xshown in Formula I. In each of the adaptor structures shown above, thenitrogen of the amine moiety can also be a component of the linker(e.g., derived from the amine moiety of an amino acid). In thoseembodiments in which an oxygen atom is shown, this oxygen atom can bebound to a phosphorus atom of a nucleic acid such as shown in FormulaII. In various embodiments, an amine moiety of the linker isfunctionalized with a C₁-C₁₀ alkyl moiety substituted with a hydroxylgroup. In various embodiments an amine moiety of the linker isfunctionalized with an adaptor shown above containing a hydroxyl group.In such embodiments, the hydroxyl group becomes the locus for attachingthe linker and the phosphorus atom of the nucleic acid, forming a P—Obond.

In various embodiments, the invention provides nucleic acid analoguesaccording to the formula:

In which L¹, L², R, Y and e are as described above, and e is an integerselected from 1, 2, 3, 4, 5 or greater. In an exemplary embodiment, L¹and or L² is or includes an adaptor. R is an amino acid side chain or asubstituted amino acid side chain. In an exemplary embodiment, thelinker includes at least one, at least two, at least three or moresulfocysteine moieties.

In an exemplary embodiment, the dyes conjugated into compounds of theinvention are fluorescent cyanines Exemplary cyanine dyes in thecompounds of the invention have the formula:

A and B independently selected monocyclic, bicyclic or polycyclic arylor heteroaryl moieties. When A and/or B is a bicyclic polycyclic moiety,two or more of the rings are optionally fused. Exemplary polycyclicmoieties include indole and benzoindole. Q is a substituted orunsubstituted methine moiety (e.g., —(CH═C(R))_(c)—CH═), in which c isan integer selected from 1, 2, 3, 4, or 5 and R is an “alkyl groupsubstituent” as defined herein. When two or more R groups are present,they are optionally joined to form a ring. Each R^(w), R^(x), R^(y) andR^(z) is independently selected from those substituents set forth in theDefinitions section herein as “alkyl group substituents” and “aryl groupsubstituents.” The indices w and z are independently selected from theintegers from 0 to 6. In an exemplary embodiment, at least one of R^(w),R^(x), R^(y) and R^(z) is C(O)NR^(o)(CH₂)_(h)G in which G is a memberselected from SO₃H and CO₂H, R^(o) is H or substituted or unsubstitutedalkyl or heteroalkyl and the index h is an integer from 1 to 20. Inexemplary embodiments, at least 1, 2, 3, 4, 5, or 6 of R^(x), R^(y),R^(w) and R^(z) are alkylsulfonic acid or heteroalkylsulfonic acid andat least one of these moieties is alkylcarboxylic acid orheteroalkylcarboxylic acid. In exemplary embodiments, at least one ofR^(w), R^(x), R^(y) and R^(z) includes a water-soluble polymer (e.g.,poly(ethylene glycol)) component. At least one of R^(x), R^(y), R^(w)and R^(z) is (L¹)_(a)-(AA)_(n)-(L²)-X as this species is defined herein.

Exemplary cyanine dyes of use in forming the compounds of the inventionare set forth in commonly owned U.S. Provisional Patent Application Nos.61/377,048, bearing attorney docket number 067191-5037PR, titled“Cyanine Dyes”, 61/377,038, bearing attorney docket number067191-5038PR, titled “Assymetric Cyanine Dyes”, 61/377,022 bearingattorney docket number 067191-5040PR, titled, “Scaffold-Based Dyes”, and61/377,004 bearing attorney docket number 067191-5041PR, titled,“Molecular Adaptors for Dye Conjugates”. The disclosure of each of theseapplications is incorporated herein by reference in its entirety for allpurposes.

Synthesis

Exemplary modes of synthesizing the compounds of the invention are setforth in the schemes below.

In Scheme 1, R is H or a substituted or unsubstituted amino acidside-chain. An exemplary substituted amino acid side chain issubstituted with a phosphate moiety (Scheme 4). The index n is selectedfrom the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and higher.The index 1 represents an integer selected from 0, 1, 2, 3, 4, 5, 6 andhigher.

In an exemplary embodiment, illustrated by Scheme 1, amino acid 1 isN-protected and converted to N-hyrdoxysuccinimide ester 3. A secondamino acid, either the same as or different from the first amino acid iscoupled to the N-protected first amino acid, forming dipeptide 4. In asimilar process, the dipeptide is coupled to a third amino acid, whichis the same as either the first or second amino acid or different fromboth of these amino acids, forming tripeptide 7. This process continuesuntil a peptide of the desired length and sequence is formed. There isno practical limitation on the sequence or length of peptides of use inthe conjugates of the invention. Exemplary peptides include at least 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

The peptide linker is coupled to L-NA, linker-nucleic acid (e.g.,polyphosphate) cassette 9 to form fluorescent nucleic acid analogue 10.Exemplary linkers, L, of use in the conjugates of the invention includesubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl moieties.

In Scheme 2, protected, activated lysine derivative 11 is reacted withnucleic acid linker cassette 12 to form conjugate 13. A secondprotected, activated lysine derivative is reacted with 13, formingdipeptide 14, which is reacted with 11, forming tri-lysyl peptide 15.The amino acid and peptide are conjugated to a dye to form 16, 17 and18.

In Scheme 3, protected, activated glutamic acid derivative 19 is reactedwith linker nucleic acid cassette 12 to form 20. Compound 20 is reactedwith 19 to form dipeptide 21, which is reacted with 19 to formtripeptide 22. The amino acid or peptide is reacted with a dye to form23, 24 or 25.

In Scheme 4, protected phosphoserine derivative 1 is converted tocorresponding NHS ester 2 and coupled to nucleic acid linker cassette 3,forming conjugate 4. The amino acid is converted to dipeptide 5 by theaction of 2. The dipeptide is converted to tripeptide 6 by addition of2. The amino acid or peptide is deprotected and coupled to a dye,forming 7, 8 or 9.

As is apparent from the Schemes above, in various embodiments, the dyeis bonded to the linker through an amide moiety formed by reaction of anamine moiety on a linker and an activated carboxylic acid group on thedye moiety. In an exemplary embodiment, the carboxylic acid group is asubstituent on a cyanine dye.

Reactive Functional Groups

The compounds of the invention are assembled from covalent bondingreactions between precursors bearing a reactive functional group, whichis a locus for formation of a covalent bond between the precursors. Theprecursors of compounds of the invention bear a reactive functionalgroup, which can be located at any position on the compound.

Exemplary species include a reactive functional group attached directlyto a cyanine nucleus (e.g., aryl ring or methine bridge) or to a linkerattached to a component (e.g., aryl ring or methine bridge) of the dyemolecule. Other molecules include a reactive functional group attachedto a polyvalent moiety. An exemplary reactive functional group isattached to an alkyl or heteroalkyl moiety on the compound. When thereactive group is attached a substituted or unsubstituted alkyl orsubstituted or unsubstituted heteroalkyl linker moiety, the reactivegroup is preferably located at a terminal position of the alkyl orheteroalkyl chain. Reactive groups and classes of reactions useful inpracticing the present invention are generally those that are well knownin the art of bioconjugate chemistry. Currently favored classes ofreactions available with reactive dye-based compounds of the inventionare those proceeding under relatively mild conditions. These include,but are not limited to nucleophilic substitutions (e.g., reactions ofamines and alcohols with acyl halides, active esters), electrophilicsubstitutions (e.g., enamine reactions) and additions to carbon-carbonand carbon-heteroatom multiple bonds (e.g., Michael reaction,Diels-Alder addition). These and other useful reactions are discussedin, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley& Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, AcademicPress, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS;Advances in Chemistry Series, Vol. 198, American Chemical Society,Washington, D.C., 1982.

Useful reactive functional groups include, for example:

-   -   (a) carboxyl groups and derivatives thereof including, but not        limited to activated esters, e.g., N-hydroxysuccinimide esters,        N-hydroxyphthalimide, N-hydroxybenztriazole esters, acid        halides, acyl imidazoles, thioesters, p-nitrophenyl esters,        alkyl, alkenyl, alkynyl and aromatic esters, activating groups        used in peptide synthesis and acid halides;    -   (b) hydroxyl groups, which can be converted to esters,        sulfonates, phosphoramidates, ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be displaced with a        nucleophilic group such as, for example, an amine, a carboxylate        anion, thiol anion, carbanion, or an alkoxide ion, thereby        resulting in the covalent attachment of a new group at the site        of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, allowing derivatization via        formation of carbonyl derivatives, e.g., imines, hydrazones,        semicarbazones or oximes, or via such mechanisms as Grignard        addition or alkyllithium addition;    -   (f) sulfonyl halide groups for reaction with amines, for        example, to form sulfonamides;    -   (g) thiol groups, which can be converted to disulfides or        reacted with acyl halides, for example;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds; and    -   (k) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assembleor utilize the reactive dye analogue. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In addition to those embodiments in which a compound of the invention isattached directly to a carrier molecule, the fluorophores can also beattached by indirect means. In this embodiment, a ligand molecule (e.g.,biotin) is generally covalently bound to the probe species. The ligandthen binds to another molecules (e.g., streptavidin) molecule, which iseither inherently detectable or covalently bound to a signal system,such as a fluorescent compound, or an enzyme that produces a fluorescentcompound by conversion of a non-fluorescent compound. Useful enzymes ofinterest as labels include, for example, hydrolases, particularlyphosphatases, esterases and glycosidases, hydrolases, peptidases oroxidases, and peroxidases.

Polyphosphate Analogues

In an exemplary embodiment, the present invention is generally directedto compositions that comprise compounds analogous to nucleotides, andwhich, in various aspects are readily processible by nucleic acidprocessing enzymes, such as polymerases. In addition to the unexpectedlyadvantageous features imparted to the compounds by incorporation of dyesof novel structure, the compounds of the invention generally benefitfrom one or more advantages of greater stability to undesired enzymaticor other cleavage or non-specific degradation, as well as incorporationefficiencies that are better than or at least comparable totriphosphate, tetraphosphate or pentaphosphate analogs.

In various embodiments, the invention provides polyphosphate analogs ofthe dyes of the invention. In various embodiments, the polyphosphateanalogs are polyphosphate analogue of a nucleic acid. An exemplarycompound according to this motif has the general structure:

in which NA is the nucleic acid. The index u is an integer selected from1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In an exemplary embodiment, the polyphosphate analogue of the inventionhas the general structure:

in which Y is a naturally occurring or non-natural nucleobase, and thelinker includes an amino acid or peptide.

In various embodiments, the polyphosphate analogue of the invention hasthe general structure:

in which t is an integer selected from 1-40, more particularly, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or higher.The index n is an integer selected from 1 to 20, for example 1, 2, 3, 4,5, 6, 7, 8, 9 or 10.

In an exemplary embodiment, the polyphosphate analogue of the inventionhas the general structure:

As will be apparent to those of skill in the art, the component labeleddye can be a cyanine.

In an exemplary embodiment, the dye component comprises multiple dyesbound to a common polyvalent scaffold or amplifier. Examples of suchscaffold-based dyes are described in commonly owned U.S. ProvisionalPatent Application No. 61/377,022, bearing Attorney Docket No.67191-5040, the disclosure of which is incorporated in its entiretyherein by reference for all purposes. The scaffold-based dyes of theinvention can include FET or FRET pairs. In an exemplary embodiment, thescaffold-based dye composition includes a Cy3 and a Cy5 type of dye(e.g., of the current invention) attached to a common polyvalentscaffold or amplifier.

In various embodiments, the polyvalent moiety is a residue of a parentcompound bound to one or more conjugated moiety, e.g., fluorescent dyemoiety, one or more peptide linker-fluorescent dye moiety cassette, oneor more nucleic acid and/or one or more linker-nucleic acid cassette. Anexemplary nucleic acid of use in these embodiments is a polyphosphatemoiety. Exemplary fluorescent dyes are cyanine dyes as disclosed hereinand in documents incorporated herein by reference.

Exemplary parent compounds for polyvalent moieties include, for example,X is a residue derived from a member selected from triazine, perylene,piperidine, phenylalanine, diaminopropanoic acid, aspartic acid, lysine,glutamic acid, serine, aminoadipic acid, 3,5-dihydroxybenzoic acid,2-amino-4-hydroxy-butyric acid, 4-(1-amino-1-carboxyethyl)-benzoic acid,piperazine-2-carboxylic acid,4-[4,6-bis-(piperidin-4-ylamino)-[1,3,5]triazin-2-ylamino]-cyclohexanecarboxylicacid and 3-amino-3-[4-(3-amino-prop-1-ynyl)-phenyl]-propionic acid.

Those of skill in the art appreciate that, in various embodiments, theparent compound is converted to the polyvalent moiety by reaction of areactive functional group on the parent compound with a reactivefunctional group on a conjugated moiety, thereby forming a covalent bondbetween the two reaction partners.

Probes

The invention provides probes having a dye of the invention conjugatedto a carrier molecule, for example, a target species (e.g., receptor,enzyme, etc.) a ligand for a target species (e.g., nucleic acid,peptide, etc.), a small molecule (e.g., drug, pesticide, etc.), a solidsupport and the like. The probes can be used for in vitro and in vivoapplications. Exemplary probes are those in which the dye is conjugatedto the carrier molecule through an adaptor or through a linker-adaptorcassette.

Small Molecule Probes

The dyes of the invention can be used as components of small moleculeprobes. In a preferred design, a small molecule probe includes a dye ofthe invention and a second species that alters the luminescentproperties of the dyes, e.g., a quencher of fluorescence. In anexemplary embodiment, an agent, such as an enzyme cleaves the dye of theinvention, the quencher or both from the small molecule generatingfluorescence in the system under investigation (see, for example,Zlokarnik et al., Science 279: 84-88 (1998)).

Nucleic Acid Capture Probes

In one embodiment, an immobilized nucleic acid comprising a dye of theinvention is used as a capture probe. The nucleic acid probe can be usedin solution phase or it can be attached to a solid support. Theimmobilized probes can be attached directly to the solid support orthrough a linker arm between the support and the dye or between thesupport and a nucleic acid residue. Preferably, the probe is attached tothe solid support by a linker (i.e., spacer arm, supra). The linkerserves to distance the probe from the solid support. The linker is mostpreferably from about 5 to about 30 atoms in length, more preferablyfrom about 10 to about 50 atoms in length. Exemplary attachment pointsinclude the 3′- or 5′-terminal nucleotide of the probe as well as otheraccessible sites discussed herein.

Chemical synthesis of nucleic acid probes containing a dye of theinvention is optionally automated and is performed by couplingnucleosides through phosphorus-containing covalent linkages. The mostcommonly used oligonucleotide synthesis method involves reacting anucleoside with a protected cyanoethyl phosphoramidite monomer in thepresence of a weak acid. The coupling step is followed by oxidation ofthe resulting phosphite linkage. Finally, the cyanoethyl protectinggroup is removed and the nucleic acid is cleaved from the solid supporton which it was synthesized. The labels of the present invention can beincorporated during oligonucleotide synthesis using a mono- orbis-phosphoramidite derivative of the fluorescent compound of theinvention. Alternatively, the label can be introduced by combining acompound of the invention that includes a reactive functional group withthe nucleic acid under appropriate conditions to couple the compound tothe nucleic acid. In yet another embodiment, the fluorescent compound isattached to a solid support through a linker arm, such as a substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl or anucleic acid residue. Synthesis proceeds with the fluorescent moietyalready in place on the growing nucleic acid chain.

Enzymatic methods of synthesis involve the use of fluorescent-labelednucleic acids in conjunction with a nucleic acid template, a primer andan enzyme. Efficient enzymatic incorporation of a fluorescent-labelednucleic acid is facilitated by selection of reaction partners that donot adversely affect the enzymes ability to couple the partners.

In those embodiments of the invention in which the dye-based fluorescentcompound of the invention is attached to a nucleic acid, the carriermolecule is produced by either synthetic (solid phase, liquid phase or acombination) or enzymatically or by a combination of these processes.

Another synthetic strategy for the preparation of oligonucleotides isthe H-phosphonate method (B. Froehler and M. Matteucci, TetrahedronLett., vol 27, p 469-472, 1986). This method utilizes activatednucleoside H-phosphonate monomers rather than phosphoramidites to createthe phosphate internucleotide linkage. In contrast to thephosphoramidite method, the resulting phosphonate linkage does notrequire oxidation every cycle but instead only a single oxidation stepat the end of chain assembly. The H-phosphonate method may also be usedto conjugate reporters and dyes to synthetic oligonucleotide chains (N.Sinha and R. Cook, Nucleic Acids Research, Vol 16, p. 2659, 1988).

In an exemplary embodiment, the synthesis and purification of thenucleic acid conjugates of compounds of the invention results in ahighly pure conjugate, which, if it is a mixture, less than about 30% ofthe nucleic acid is unlabeled with a dye of the invention, preferablyless than about 20% are unlabeled, more preferably less than about 10%,still more preferably less than about 5%, more preferably less thanabout 1%, more preferably less than about 0.5%, or more preferably lessthan about 0.1% and even more preferably less than 0.01% of the nucleicacid is unlabeled with a dye of the invention. In certain embodiments,the nucleic acid (e.g., nucleotides and/or nucleotide analogs) isincorporatable by a polymerase enzyme in a template-dependentpolymerization reaction.

Dual Labeled Probes

The present invention also provides dual labeled probes that includeboth a dye of the invention and another label. Exemplary dual labeledprobes include nucleic acid probes that include a nucleic acid with adye of the invention attached thereto. Exemplary probes include both adye of the invention and a quencher. The probes are of use in a varietyof assay formats. For example, when a nucleic acid singly labeled with adye of the invention is the probe, the interaction between the first andsecond nucleic acids can be detected by observing the interactionbetween the dye of the invention and the nucleic acid. Alternatively,the interaction is the quenching by a quencher attached to the secondnucleic acid of the fluorescence from a dye of the invention.

The dyes of the invention are useful in conjunction with nucleic-acidprobes in a variety of nucleic acid amplification/quantificationstrategies including, for example, 5′-nuclease assay, StrandDisplacement Amplification (SDA), Nucleic Acid Sequence-BasedAmplification (NASBA), Rolling Circle Amplification (RCA), as well asfor direct detection of targets in solution phase or solid phase (e.g.,array) assays. Furthermore, the dye of the invention-derivatized nucleicacids can be used in probes of substantially any format, including, forexample, format selected from molecular beacons, Scorpion Probes™,Sunrise Probes™, conformationally assisted probes, light up probes,Invader Detection probes, and TaqMan™ probes. See, for example,Cardullo, R., et al., Proc. Natl. Acad. Sci. USA, 85:8790-8794 (1988);Dexter, D. L., J. Chem. Physics, 21:836-850 (1953); Hochstrasser, R. A.,et al., Biophysical Chemistry, 45:133-141 (1992); Selvin, P., Methods inEnzymology, 246:300-334 (1995); Steinberg, I., Ann. Rev. Biochem.,40:83-114 (1971); Stryer, L., Ann. Rev. Biochem., 47:819-846 (1978);Wang, G., et al., Tetrahedron Letters, 31:6493-6496 (1990); Wang, Y., etal., Anal. Chem., 67:1197-1203 (1995); Debouck, C., et al., insupplement to nature genetics, 21:48-50 (1999); Rehman, F. N., et al.,Nucleic Acids Research, 27:649-655 (1999); Cooper, J. P., et al.,Biochemistry, 29:9261-9268 (1990); Gibson, E. M., et al., GenomeMethods, 6:995-1001 (1996); Hochstrasser, R. A., et al., BiophysicalChemistry, 45:133-141 (1992); Holland, P. M., et al., Proc Natl. Acad.Sci USA, 88:7276-7289 (1991); Lee, L. G., et al., Nucleic Acids Rsch.,21:3761-3766 (1993); Livak, K. J., et al., PCR Methods and Applications,Cold Spring Harbor Press (1995); Vamosi, G., et al., BiophysicalJournal, 71:972-994 (1996); Wittwer, C. T., et al., Biotechniques,22:176-181 (1997); Wittwer, C. T., et al., Biotechniques, 22:130-38(1997); Giesendorf, B. A. J., et al., Clinical Chemistry, 44:482-486(1998); Kostrikis, L. G., et al., Science, 279:1228-1229 (1998); Matsuo,T., Biochemica et Biophysica Acta, 1379:178-184 (1998); Piatek, A. S.,et al., Nature Biotechnology, 16:359-363 (1998); Schofield, P., et al.,Appl. Environ. Microbiology, 63:1143-1147 (1997); Tyagi S., et al.,Nature Biotechnology, 16:49-53 (1998); Tyagi, S., et al., NatureBiotechnology, 14:303-308 (1996); Nazarenko, I. A., et al., NucleicAcids Research, 25:2516-2521 (1997); Uehara, H., et al., Biotechniques,26:552-558 (1999); D. Whitcombe, et al., Nature Biotechnology,17:804-807 (1999); Lyamichev, V., et al., Nature Biotechnology, 17:292(1999); Daubendiek, et al., Nature Biotechnology, 15:273-277 (1997);Lizardi, P. M., et al., Nature Genetics, 19:225-232 (1998); Walker, G.,et al., Nucleic Acids Res., 20:1691-1696 (1992); Walker, G. T., et al.,Clinical Chemistry, 42:9-13 (1996); and Compton, J., Nature, 350:91-92(1991).

In view of the well-developed body of literature concerning theconjugation of small molecules to nucleic acids, many other methods ofattaching donor/acceptor pairs to nucleic acids will be apparent tothose of skill in the art.

More specifically, there are many linking moieties and methodologies forattaching groups to the 5′- or 3′-termini of nucleic acids, asexemplified by the following references: Eckstein, editor, Nucleic acidsand Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckermanet al., Nucleic Acids Research, 15: 5305-5321 (1987) (3′-thiol group onnucleic acid); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′-sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′-phosphoamino groupvia Aminolink TM II available from P.E. Biosystems, CA.) Stabinsky, U.S.Pat. No. 4,739,044 (3-aminoalkylphosphoryl group); Agrawal et al.,Tetrahedron Letters, 31: 1543-1546 (1990) (attachment viaphosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5-mercapto group); Nelson et al., Nucleic Acids Research,17: 7187-7194 (1989) (3′-amino group), and the like.

Exemplary fluorophores that can be combined in a probe or scaffold-baseddye with a dye of the invention include those set forth in Table 1.

TABLE 1 Exemplary Donors or Acceptors for Compounds of the Invention4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives:   acridine   acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonateN-(4-anilino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant Yellowcoumarin and derivatives: coumarin   7-amino-4-methylcoumarin (AMC,Coumarin 120)   7-amino-4-trifluoromethylcouluarin (Coumaran 151)cyanine dyes cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives:   eosin   eosin isothiocyanate erythrosin and derivatives:  erythrosin B   erythrosin isothiocyanate ethidium fluorescein andderivatives:   5-carboxyfluorescein (FAM)  5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)  2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)   fluorescein  fluorescein isothiocyanate   QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives:   pyrene butyrate  succinimidyl 1-pyrene butyrate quantum dots Reactive Red 4 (Cibacron ™Brilliant Red 3B-A) rhodamine and derivatives:   6-carboxy-X-rhodamine(ROX)   6-carboxyrhodamine (R6G)   lissamine rhodamine B sulfonylchloride rhodamine (Rhod)   rhodamine B   rhodamine 123   rhodamine Xisothiocyanate   sulforhodamine B   sulforhodamine 101 sulfonyl chloridederivative of sulforhodamine 101 (Texas Red)N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine  tetramethyl rhodamine isothiocyanate (TRITC) riboflavin rosolic acidterbium chelate derivatives Black Hole Quenchers ™

There is a great deal of practical guidance available in the literaturefor functionalizing fluorophores and selecting appropriatedonor-acceptor pairs for particular probes, as exemplified by thefollowing references: Pesce et al., Eds., FLUORESCENCE SPECTROSCOPY(Marcel Dekker, New York, 1971); White et al., FLUORESCENCE ANALYSIS: APRACTICAL APPROACH (Marcel Dekker, New York, 1970); and the like. Theliterature also includes references providing exhaustive lists offluorescent and chromogenic molecules and their relevant opticalproperties for choosing reporter-quencher pairs (see, for example,Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2ndEdition (Academic Press, New York, 1971); Griffiths, COLOUR ANDCONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976);Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes,Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing reporter and quenchermolecules for covalent attachment via common reactive groups that can beadded to a nucleic acid, as exemplified by the following references:Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al.,U.S. Pat. No. 4,351,760. Thus, it is well within the abilities of thoseof skill in the art to choose an energy exchange pair for a particularapplication and to conjugate the members of this pair to a probemolecule, such as, for example, a nucleic acid, peptide or otherpolymer.

As will be apparent to those of skill in the art the methods set forthabove are equally applicable to the coupling to a nucleic acid of groupsother than the fluorescent compounds of the invention, e.g., quenchers,intercalating agents, hybridization enhancing moieties, minor groovebinders, alkylating agents, cleaving agents, etc.

When the nucleic acids are synthesized utilizing an automated nucleicacid synthesizer, the donor and acceptor moieties are preferablyintroduced during automated synthesis. Alternatively, one or more ofthese moieties can be introduced either before or after the automatedsynthesis procedure has commenced. For example, donor and/or acceptorgroups can be introduced at the 3′-terminus using a solid supportmodified with the desired group(s). Additionally, donor and/or acceptorgroups can be introduced at the 5′-terminus by, for example a derivativeof the group that includes a phosphoramidite. In another exemplaryembodiment, one or more of the donor and/or acceptor groups isintroduced after the automated synthesis is complete.

In the dual labeled probes, the quencher moiety is preferably separatedfrom the dye of the invention by at least about 10 nucleotides, and morepreferably by at least about 15 nucleotides. The quencher moiety ispreferably attached to either the 3′- or 5′-terminal nucleotides of theprobe. The dye of the invention moiety is also preferably attached toeither the 3′- or 5′-terminal nucleotides of the probe. More preferably,the donor and acceptor moieties are attached to the 3′- and 5′- or 5′-and 3′-terminal nucleotides of the probe, respectively, althoughinternal placement is also useful.

Once the desired nucleic acid is synthesized, it is preferably cleavedfrom the solid support on which it was synthesized and treated, bymethods known in the art, to remove any protecting groups present (e.g.,60° C., 5 h, concentrated ammonia). In those embodiments in which abase-sensitive group is attached to the nucleic acids (e.g., TAMRA), thedeprotection will preferably use milder conditions (e.g., butylamine:water 1:3, 8 hours, 70° C.). Deprotection under these conditions isfacilitated by the use of quick deprotect amidites (e.g., dC-acetyl,dG-dmf).

Peptide Probes

Peptides, proteins and peptide nucleic acids that are labeled with aquencher and a dye of the invention can be used in both in vivo and invitro enzymatic assays.

Peptide constructs useful in practicing the invention include those withthe following features: i) a quencher; ii) a dye of the invention; andiii) a cleavage or assembly recognition site for the enzyme. Moreover,the peptide construct is preferably exists in at least one conformationthat allows donor-acceptor energy transfer between the dye of theinvention and the quencher when the fluorophore is excited.

In the dual labeled probes of the invention, the donor and acceptormoieties are connected through an intervening linker moiety. The linkermoiety, preferably, includes a peptide moiety, but can be or can includeanother organic molecular moiety, as well. In a preferred embodiment,the linker moiety includes a cleavage recognition site specific for anenzyme or other cleavage agent of interest. A cleavage site in thelinker moiety is useful because when a tandem construct is mixed withthe cleavage agent, the linker is a substrate for cleavage by thecleavage agent. Rupture of the linker moiety results in separation ofthe dye and the quencher. The separation is measurable as a change indonor-acceptor energy transfer. Alternatively, peptide assembly can bedetected by an increase in donor-acceptor energy transfer between apeptide fragment bearing a fluorescent dye and a peptide fragmentbearing a donor moiety.

When the cleavage agent of interest is a protease, the linker generallyincludes a peptide containing a cleavage recognition sequence for theprotease. A cleavage recognition sequence for a protease is a specificamino acid sequence recognized by the protease during proteolyticcleavage. Many protease cleavage sites are known in the art, and theseand other cleavage sites can be included in the linker moiety. See,e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994);Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol.244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); Bouvier etal. Meth. Enzymol. 248: 614 (1995), Hardy et al., in AMYLOID PROTEINPRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMER'S DISEASE, ed. Masters etal. pp. 190-198 (1994).

Solid Support Immobilized Dye Analogues

The amino acid or peptide linked dyes of the invention can beimmobilized on substantially any polymer, biomolecule, or solid orsemi-solid material having any useful configuration. Moreover, anyconjugate comprising one or more dye of the invention can be similarlyimmobilized. When the support is a solid or semi-solid, examples ofpreferred types of supports for immobilization of the nucleic acid probeinclude, but are not limited to, controlled pore glass, glass plates,polystyrene, avidin coated polystyrene beads, cellulose, nylon,acrylamide gel and activated dextran. These solid supports are preferredbecause of their chemical stability, ease of functionalization andwell-defined surface area. Solid supports such as, controlled pore glass(CPG, 500 Å, 1000 Å) and non-swelling high cross-linked polystyrene(1000 Å) are particularly preferred.

According to the present invention, the surface of a solid support isfunctionalized with a dye of the invention or a species to which a dyeof the invention is conjugated. For clarity of illustration, thefollowing discussion focuses on attaching a reactive dye of theinvention to a solid support. The following discussion is also broadlyrelevant to attaching to a solid support a species that includes withinits structure a dye of the invention.

The dyes of the invention are preferably attached to a solid support byforming a bond between a reactive group on the dye of the invention(e.g., on an amino acid or peptide linker) and a reactive group on thesurface of the solid support, thereby derivatizing the solid supportwith one or more dye of the invention. Alternatively, the reactive groupon the dye of the invention is coupled with a reactive group on a linkerarm attached to the solid support. The bond between the solid supportand the dye of the invention is preferably a covalent bond, althoughionic, dative and other such bonds are useful as well. Reactive groupswhich can be used in practicing the present invention are discussed indetail above and include, for example, amines, hydroxyl groups,carboxylic acids, carboxylic acid derivatives, alkenes, sulfhydryls,siloxanes, etc.

A large number of solid supports appropriate for practicing the presentinvention are available commercially and include, for example, peptidesynthesis resins, both with and without attached amino acids and/orpeptides (e.g., alkoxybenzyl alcohol resin, aminomethyl resin,aminopolystyrene resin, benzhydrylamine resin, etc. (Bachem)),functionalized controlled pore glass (BioSearch Technologies, Inc.), ionexchange media (Aldrich), functionalized membranes (e.g., —COOHmembranes; Asahi Chemical Co., Asahi Glass Co., and Tokuyama Soda Co.),and the like.

Microarrays

The present invention also provides microarrays including immobilizeddye of the invention and compounds (e.g., peptides, nucleic acids,bioactive agents, etc.) functionalized with a dye of the invention.Moreover, the invention provides methods of interrogating microarraysusing probes that are functionalized with a dye of the invention. Theimmobilized species and the probes are selected from substantially anytype of molecule, including, but not limited to, small molecules,peptides, enzymes nucleic acids and the like.

Nucleic acid microarrays consisting of a multitude of immobilizednucleic acids are revolutionary tools for the generation of genomicinformation, see, Debouck et al., in supplement to Nature Genetics,21:48-50 (1999). The discussion that follows focuses on the use of a dyeof the invention in conjunction with nucleic acid microarrays. Thisfocus is intended to be illustrative and does not limit the scope ofmaterials with which this aspect of the present invention can bepracticed. See, Lehrach, et al., HYBRIDIZATION FINGERPRINTING IN GENOMEMAPPING AND SEQUENCING, GENOME ANALYSIS, Vol. 1, Davies et al, Eds.,Cold Springs Harbor Press, pp. 39-81 (1990), Pirrung et al. (U.S. Pat.No. 5,143,854, issued 1992), and also by Fodor et al., (Science, 251:767-773 (1991), Southern et al. (Genomics, 13: 1008-1017 (1992),Khrapko, et al., DNA Sequence, 1: 375-388 (1991), Kleinfield et al., J.Neurosci. 8:4098-120 (1998)), Kumar et al., Langmuir 10:1498-511 (1994),Xia, Y., J. Am. Chem. Soc. 117:3274-75 (1995), Hickman et al., J. Vac.Sci. Technol. 12:607-16 (1994), Mrkish et al. Ann. Rev. Biophys. Biomol.Struct. 25:55-78 (1996).

Probes of Enzymatic Reactions

In various embodiments, the invention provides a composition which is asubstrate for an enzyme, the substrate comprising a component reactedupon by the enzyme, a fluorescent label component and an amino acid orpeptide linker component conjugating these two components. The linkercomponent interacts with the enzyme to increase the affinity of thefluorophore-linker-enzyme reactive component with the enzyme, reducingthe K_(m) of the reaction between the enzyme and the enzyme-reactivecomponent relative to that of an analogous reaction in which theconjugate does not include the linker component. Exemplary interactionmodalities by which the linker increases the affinity of the conjugatefor the enzyme include, without limitation, electrostatic, hydrophobicand steric interactions. In various embodiments, the K_(m) is reduced atleast 10%, at least 20%, at least 30%, at least 40% or at least 50%relative to the K_(m) of the reaction with an analogous conjugatewithout the linker component.

Probes that do not have the amino acid or peptide linker of thecompounds of the present invention are referred to as “otherwiseidentical” to the probes having the amino acid or peptide linker. Suchprobes are represented by the formula:

R¹-(L¹)_(a)-L³-(L²)_(b)-X

in which each of the radicals and indices is as discussed herein, and L³is a non-amino acid, non-peptide linker. An exemplary linker for L³ is asubstituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl linker. In various embodiments, L³ is from 1 to 12 atomslong, e.g., from 2 to 10 atoms, or from 4 to 8 atoms long. In variousembodiments, L³ is a bond (“zero order linker”) linking two componentsof the conjugate.

In various embodiments, the linker serves to enhance the interactionbetween a conjugate of the invention and a protein, such as a DNApolymerase. The linker can enhance the interaction between the conjugateand the protein through electrostatic, hydrophobic, or stericinteractions. In an exemplary embodiment in which the conjugate isutilized in a single molecule nucleic acid sequencing technique, thelinker enhances the interaction between the conjugate and the DNApolymerase, thereby lowering the K_(m) of the sequencing reaction andinfluencing the 2-slow step to achieve optimized residence time of theconjugate on the polymerase and enzyme kinetics. In examples of thisembodiment, the linker is an amino acid or peptide. In variousembodiments, the conjugate has the formula:

in which L¹ and L² are independently selected from bonds and adaptors,e.g., substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl. The index u″ is an integer selected from 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12 or higher. R^(L) is H or is an amino acid side-chain(e.g., NH₂, SH, COOH) or a derivatized side chain (e.g., CH₂OP(O)(OR)₂,wherein each R is independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted heterocycloalkyl. The indexn is an integer selected from 0, 1, 2, 3, 4, 5, 6 or higher. Each of then amino acid residues is independently selected from naturally occurringor unnatural amino acids. An exemplary amino acid/peptide linker is onein which n is an integer selected from 1, 2, 3, 4, 5 or higher. Invarious embodiments, the amino acid/peptide is lysine or a peptidecontaining lysine, glutamic acid or a peptide containing glutamic acid,serine or a peptide containing serine. In various embodiments, thepeptide linker is composed only of lysine, only of glutamic acid, onlyof serine or only of O-phosphoserine (or an ester thereof).

In various embodiments, the linker in the conjugate of the inventionincludes one, two, three or more anionic amino acids in the linker. Anexemplary linker includes only anionic amino acids. In variousembodiments, the linker in the conjugate of the invention includes one,two, three or more hydrophobic amino acids. An exemplary linker includesonly hydrophobic amino acids. In various embodiments, the linker in theconjugates of the invention includes one, two, three or more cationicamino acids in the linker. An exemplary linker includes only cationicamino acids. The various permutations of combinations of anionic,hydrophobic and cationic amino acids in a linker of 1, 2, 3, 4, 5, or 6amino acid residues is encompassed in this disclosure.

In an exemplary embodiment, the linker in the compounds of the inventionincludes at least one amino acid besides lysine. In various embodiments,the linker is not poly- or oligo-lysine. In various embodiments, thelinker is not Lys, Lys₄, Lys₅ or Lys₆. In a further exemplaryembodiment, the linker is not poly- or oligo-proline. In variousembodiments, the linker is not Pro, Pro₂, Pro₃, Pro₄, Pro₅ or Pro₆.

Additional peptide linkers of use in the present invention are set forthin commonly owned U.S. Patent Application Publication No. 20090233302,the disclosure of which is incorporated in its entirety herein byreference for all purposes.

The Methods

In addition to the compounds of the invention, there are also providedan array of methods utilizing the compounds. The following discussion isintended to be illustrative of the type and scope of methods with whichthe compounds of the invention can be practiced and should not beinterpreted as being either exhaustive or limiting.

Monitoring Enzymatic Reactions

Peptides, proteins and peptide nucleic acids that are labeled with aquencher and a dye of the invention can be used in both in vivo and invitro enzymatic assays.

Thus, in another aspect, the present invention provides a method fordetermining whether a sample contains an enzyme. The method comprises:(a) contacting the sample with a peptide construct that includes a dyeof the invention; (b) exciting the fluorophore; and (c) determining afluorescence property of the sample, wherein the presence of the enzymein the sample results in a change in the fluorescence property.

Peptide constructs useful in practicing the invention include those withthe following features: i) a quencher; ii) a dye of the invention; andiii) a cleavage or assembly recognition site for the enzyme. Moreover,the peptide construct preferably exists in at least one conformationthat allows donor-acceptor energy transfer between the dye of theinvention and the quencher when the fluorophore is excited.

The assay is useful for determining the amount of enzyme in a sample.For example, by determining the degree of donor-acceptor energy transferat a first and second time after contact between the enzyme and thetandem construct, and determining the difference in the degree ofdonor-acceptor energy transfer. The difference in the degree ofdonor-acceptor energy transfer reflects the amount of enzyme in thesample.

The assay methods also can also be used to determine whether a compoundalters the activity of an enzyme, i.e., screening assays. Thus, in afurther aspect, the invention provides methods of determining the amountof activity of an enzyme in a sample from an organism. The methodincludes: (a) contacting a sample comprising the enzyme and the compoundwith a peptide construct that includes a dye of the invention; (b)exciting the fluorophore; and (c) determining a fluorescence property ofthe sample, wherein the activity of the enzyme in the sample results ina change in the fluorescence property. Peptide constructs useful in thisaspect of the invention are substantially similar to those describedimmediately above.

In a preferred embodiment, the amount of enzyme activity in the sampleis determined as a function of the degree of donor-acceptor energytransfer in the sample and the amount of activity in the sample iscompared with a standard activity for the same amount of the enzyme. Adifference between the amount of enzyme activity in the sample and thestandard activity indicates that the compound alters the activity of theenzyme.

Representative enzymes with which the present invention can be practicedinclude, for example, nucleotide polymerases (e.g., DNA polymerase),trypsin, enterokinase, HIV-1 protease, prohormone convertase,interleukin-1b-converting enzyme, adenovirus endopeptidase,cytomegalovirus assemblin, leishmanolysin, β-secretase for amyloidprecursor protein, thrombin, renin, angiotensin-converting enzyme,cathepsin-D and a kininogenase, and proteases in general.

An exemplary assay for proteases are based on donor-acceptor energytransfer from a donor fluorophore to a quencher placed at opposite endsof a short peptide chain containing the potential cleavage site (see,Knight C. G., Methods in Enzymol. 248:18-34 (1995)). Proteolysisseparates the fluorophore and quencher, resulting in increased intensityin the emission of the donor fluorophore. Existing protease assays useshort peptide substrates incorporating unnatural chromophoric aminoacids, assembled by solid phase peptide synthesis.

In a further aspect, the invention provides a method of monitoring anenzyme reaction. The method generally comprises providing a reactionmixture comprising die enzyme and at least a first reactant composition,the reactant composition comprising a compound having a reactantcomponent, which is a substrate for the enzyme, a fluorescent labelcomponent, and a linker component joining the reactant component to thelabel component. In various embodiments, the linker component increasesthe affinity of the conjugate for the enzyme. In various embodiments,the increased affinity reduces the K_(m) of the reaction, e.g., by 10%,at least 20%, at least 30%, at least 40% or at least 50% relative to theK_(m) of the reaction with an analogous conjugate without the linkercomponent. The reaction mixture is illuminated to excite the fluorescentlabel component, and a fluorescent signal from the reaction mixturecharacteristic of the enzyme reaction is detected.

In an exemplary embodiment, the enzymatic reaction is the reaction of apolymerase with a nucleic acid.

Nucleic Acid Sequencing

In various embodiments, the present invention provides a method fornucleic acid sequencing using one or more compounds of the invention. Anexemplary sequencing method is single molecule nucleic acid sequencing.

Significant interest in the sequencing of single DNA molecules dates to1989 when Keller and colleagues began experimenting with “sequencing bydegradation.” In their experiments, isolated fully-labeled DNA moleculesare degraded by an exonuclease, and individual labeled bases aredetected as they are sequentially cleaved from the DNA (Jett, J. H. etal., J. Biomol. Struct. Dynamics, 7, 301-309 (1989); Stephan, J. et al.,J. Biotechnol., 86, 255-267 (2001); Werner, J. H. et al., J.Biotechnol., 102, 1-14 (2003)). This approach was ultimately compromisedby poor DNA solubility caused by the densely-packed dye labels. Morerecently, alternative single-molecule approaches have been investigated,including “sequencing by synthesis,” where bases are detected one at atime as they are sequentially incorporated into DNA by a polymerase(Braslaysky, I. et al., Proc. Natl. Acad. Sci. USA, 100, 3960-3964(2003); Levene, M. J. et al., Science, 299, 682-686 (2003); Metzker, M.L., Genome Res., 15, 1767-1776 (2005)); and nanopore sequencing whereelectrical signals are detected while single DNA molecules pass throughprotein or solid-state nanopores (Akeson, M. et al., Biophys. J., 77,3227-3233 (1999); Lagerqvist, J. et al., Nano Lett., 6, 779-782 (2006);Rhee, K. J. et al, Annals of emergency medicine, 13, 916-923 (1984)). Sofar, only sequencing by synthesis has been successful. In the method ofQuake and colleagues (Braslaysky, I. et al., Proc. Natl. Acad. Sci. USA,100, 3960-3964 (2003)), base-labeled nucleotide triphosphates (dNTPs)are incorporated into DNA immobilized on a microscope coverglass. Eachtype of dNTP is applied separately in a fluidics cycle, and incorporatedbases are imaged on the surface after washing away the excess of freenucleotides. While the obtained sequence reads are short, highsequencing rates can potentially be achieved by analyzing billions ofdifferent, individual molecules in parallel with applications inre-sequencing and gene expression profiling.

To obtain long single-molecule reads, potentially tens of kilobases,sequencing-by-synthesis approaches using phosphate-labeled nucleotideshave been developed (Levene, M. J. et al., Science, 299, 682-686(2003)). These nucleotides are labeled with a fluorophore on theterminal phosphate instead of on the base. Labeled nucleotides aredetected while bound to polymerase during the catalytic reaction. Thelabel is released with pyrophosphate as the nucleotide is incorporatedinto DNA. An advantage is that the DNA remains label-free and fullysoluble. Individual polymerase enzymes immobilized on a microscopecoverglass are monitored in real time to detect the sequence ofincorporated nucleotides. In order to achieve long reads, thepolymerase, but not the DNA, can be attached to the coverglass.Polymerase attachment facilitates detection because it keeps the activesite at a single position on the coverglass surface. In the alternativeformat, with the polymerase in solution and the DNA attached, the enzymeactive site would be a moving target for detection, diffusing up toseveral microns from the DNA attachment point as the primer strand isextended from long templates.

U.S. Pat. No. 6,255,083, issued to Williams and incorporated herein byreference, discloses a single molecule sequencing method on a solidsupport. The solid support is optionally housed in a flow chamber havingan inlet and outlet to allow for renewal of reactants that flow past theimmobilized polymerases. The flow chamber can be made of plastic orglass and should either be open or transparent in the plane viewed bythe microscope or optical reader.

Accordingly, it is within the scope of the present invention to utilizethe compounds set forth herein in single molecule DNA sequencing.

In accordance with one embodiment of the methods of invention, thecompounds described herein are used in analyzing nucleic acid sequencesusing a template dependent polymerization reaction to monitor thetemplate dependent incorporation of specific analogs into a synthesizednucleic acid strand, and thus determine the sequence of nucleotidespresent in the template nucleic acid strand. In particular, a polymeraseenzyme is complexed with the template strand in the presence of one ormore nucleotides and/or one or more nucleotide analogs of the invention.In preferred aspects, only the labeled analogs of the invention arepresent representing analogous compounds to each of the four naturalnucleotides, A, T, G and C. When a particular base in the templatestrand is encountered by the polymerase during the polymerizationreaction, it complexes with an available analog that is complementary tosuch nucleotide, and incorporates that analog into the nascent andgrowing nucleic acid strand, cleaving between the α and β phosphorusatoms in the analog, and consequently releasing the labeling group (or aportion thereof). The incorporation event is detected, either by virtueof a longer presence of the analog in the complex, or by virtue ofrelease of the label group into the surrounding medium. Where differentlabeling groups are used for each of the types of analogs, e.g., A, T, Gor C, identification of a label of an incorporated analog allowsidentification of that analog and consequently, determination of thecomplementary nucleotide in the template strand being processed at thattime. Sequential reaction and monitoring permits a real-time monitoringof the polymerization reaction and determination of the sequence of thetemplate nucleic acid. As noted above, in particularly preferredaspects, the polymerase enzyme/template complex is provided immobilizedwithin an optical confinement that permits observation of an individualcomplex, e.g., a zero mode waveguide. In addition to their use insequencing, the analogs of the invention are also equally useful in avariety of other genotyping analyses, e.g., SNP genotyping use singlebase extension methods, real time monitoring of amplification, e.g.,RT-PCR methods, and the like. See, for example, U.S. Pat. Nos.7,056,661, 7,052,847, 7,033,764, 7,056,676, 6,917,726, 7,013,054,7,181,122, 7,292,742 and 7,170,050 and 7,302,146, the full disclosuresof which are incorporated herein by reference in their entirety for allpurposes.

The present invention also provides methods of using the compoundsdescribed herein in performing nucleic acid analyses, and particularlynucleic acid sequence analyses. The methods of the invention typicallycomprise providing a template nucleic acid complexed with a polymeraseenzyme in a template dependent polymerization reaction to produce anascent nucleic acid strand, contacting the polymerase and templatenucleic acid with a compound of the invention, and detecting whether ornot a synthon derived from the compound (e.g., monophosphate nucleicacid subunit) was incorporated into the nascent strand during thepolymerization reaction, and identifying a base in the template strandbased upon incorporation of the compound. Preferably, the foregoingprocess is carried out so as to permit observation of individualnucleotide incorporation reactions, through the use of, for example, anoptical confinement, that allows observation of an individual polymeraseenzyme, or through the use of a heterogeneous assay system, where labelgroups released from incorporated analogs are detected.

The invention also provides methods of monitoring nucleic acid synthesisreactions. The methods comprise contacting a polymerase/template/primercomplex with a fluorescently labeled nucleotide or nucleotide analoghaving a nucleotide or nucleotide analog component, a fluorescent labelcomponent, and a linker component joining die nucleotide or nucleotideanalog component to the label component, wherein the linker componentincreases the affinity of the conjugate for the enzyme, reducing theK_(m) of the reaction, e.g., by 10%, at least 20%, at least 30%, atleast 40% or at least 50% relative to the K_(m) of the reaction with ananalogous conjugate without the linker component. A characteristicsignal from the fluorescent dye is then detected that is indicative ofincorporation of the nucleotide or nucleotide analog into a primerextension reaction.

The amino acid or peptide linked fluorophores of the invention are ofuse in single molecule or single molecule real time (SMRT) DNAsequencing assays. Of particular note in this context is the abilityprovided by the invention to design fluorophores with selectedabsorbance and emission properties including wavelength and intensity.The compounds of the invention provide for very versatile assay design.For example, according to the present invention a series of fluorophoresof use in an assay are readily designed to have selected absorbance andemission wavelengths and emission intensities, allowing multiplefluorophores to be utilized and distinguished in an assay. In exemplaryembodiments, use of compounds of the invention in a multifluorophoreassay, e.g., single molecule DNA sequencing, enhances assay performanceby at least about 10%, at least about 20% or at least about 30% over asimilar assay using currently available fluorophores.

In single-molecule DNA sequencing by synthesis, for example as describedEid, J. et al., Science, 323(5910), 133-138 (2009), the incorporation ofspecific nucleotides can be determined by observing bright phases anddark phases which correspond, for example, to reaction steps in which afluorescent label is associated with the polymerase enzyme, and steps inwhich the fluorescent label is not associated with the enzyme. In someembodiments of the invention, the polymerase reaction system willexhibit two slow (kinetically observable) reaction steps wherein each ofthe steps is in a bright phase. In some embodiments of the invention,the system will exhibit two kinetically observable reaction stepswherein each of the steps is in a dark phase. In some cases, the systemwill have four kinetically observable (slow) reaction steps, two slowsteps in a bright phase and two slow steps in a dark phase.

In an exemplary embodiment, the conjugates of the invention exhibit anenhanced two-slow step character in single molecule DNA sequencing whencompared to analogous cyanine dyes without amino acid linkers betweenthe dye and the nucleic acid. Exemplary two slow step results for acyanine dye with a lysine linker and a triphosphate nucleic acid are setforth below:

Conjugate K1 (S-1) K2 (S-1) 2SS characteristics Tri-phosphate 9.4960.371 25.6 1Lys-tri-phosphate 12.96 0.96 13.5 2Lys-tri-phosphate 2.6450.406 6.5 2Glu-tri-phosphate 7.898 1.082 7.3

Polymerase Chain Reaction

In another aspect, the invention provides a method for detectingamplification by PCR of a target sequence. Methods of monitoring PCRusing dual labeled nucleic acid probes are known in the art. See, ExpertRev. Mol. Diagn., 5(2), 209-219 (2005).

The dyes and their conjugates described herein can be used insubstantially any nucleic acid probe format. For example, the dyes ofthe invention can be incorporated into probe motifs, such as Taqman™probes (Held et al., Genome Res. 6: 986-994 (1996), Holland et al.,Proc. Nat. Acad. Sci. USA 88: 7276-7280 (1991), Lee et al., NucleicAcids Res. 21: 3761-3766 (1993)), molecular beacons (Tyagi et al.,Nature Biotechnology 14:303-308 (1996), Jayasena et al., U.S. Pat. No.5,989,823, issued Nov. 23, 1999)) scorpion probes (Whitcomb et al.,Nature Biotechnology 17: 804-807 (1999)), sunrise probes (Nazarenko etal., Nucleic Acids Res. 25: 2516-2521 (1997)), conformationally assistedprobes (Cook, R., copending and commonly assigned U.S. patentapplication Ser. No. 09/591,185), peptide nucleic acid (PNA)-based lightup probes (Kubista et al., WO 97/45539, December 1997), double-strandspecific DNA dyes (Higuchi et al, Bio/Technology 10: 413-417 (1992),Wittwer et al, BioTechniques 22: 130-138 (1997)) and the like. These andother probe motifs with which the present dyes can be used are reviewedin NONISOTOPIC DNA PROBE TECHNIQUES, Academic Press, Inc. 1992.

Nucleic Acid Detection

In another embodiment, the invention provides a method of detecting atarget nucleic acid in an assay mixture or other sample. The followingdiscussion is generally relevant to the assays described herein. Thisdiscussion is intended to illustrate the invention by reference tocertain preferred embodiments and should not be interpreted as limitingthe scope of probes and assay types in which the compounds of theinvention find use. Other assay formats utilizing the compounds of theinvention will be apparent to those of skill in the art.

An exemplary method uses a dye of the invention or a conjugate thereofto detect a nucleic acid target sequence. The method includes: (a)contacting the target sequence with a detector nucleic acid thatincludes a dye of the invention and a quencher; (b) hybridizing thedetector nucleic acid to the target sequence, thereby altering theconformation of the detector nucleic acid, causing a change in afluorescence parameter; and (c) detecting the change in the fluorescenceparameter, thereby detecting the nucleic acid target sequence.

In various embodiments, the detector nucleic acid includes asingle-stranded target binding sequence. The binding sequence has linkedthereto: i) a quencher; and ii) a dye of the invention. Moreover, priorto its hybridization to a complementary sequence, the detector nucleicacid is preferably in a conformation that allows donor-acceptor energytransfer between the quencher and the dye of the invention when thefluorophore is excited. Furthermore, in the methods described in thissection, a change in fluorescence is detected as an indication of thepresence of the target sequence. The change in fluorescence ispreferably detected in real time.

Kits

In another aspect, the present invention provides kits containing one ormore dye of the invention or a conjugate thereof. In one embodiment, akit includes a reactive dye of the invention and directions forattaching this derivative to another molecule. In another embodiment,the kit includes a dye-labeled polyphosphate nucleic acid in which anamino acid or peptide linker is present between the dye and thepolyphosphate nucleic acid. The kit further includes one or morecomponent selected from buffers or other compounds or solutions of usein practicing the method, an enzyme (e.g., a DNA polymerase), cofactorsnecessary for enzyme reactions, and directions for performing the assay.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

EXAMPLES Example 1

6-Hydrazino-1,3-naphthalenedisulfonate (1)

To a solution of disodium 6-amino-1,3-naphthalenedisulfonate hydrate(10.0 g, 28.8 mmol) in 50% hydrochloric acid (200 mL) at 0° C. was addeddropwise a cold solution of sodium nitroxide (2.18 g, 31.7 mmol) inwater (20 mL). After completion of addition (˜25 min) the solution wasstirred for an additional 30 min at 0° C. followed by dropwise additionof a cold solution of SnCl₂ (6.0 g, 31.7 mmol) in hydrochloric acid (10mL) in 40 min. Continue to stir at 0° C. for 1 h and then ambienttemperature for 1 h. Concentrated to dryness and triturated with hotiPrOH (400 mL). Filtered to collect the solid, washed with iPrOH (2×30mL), ethyl acetate (2×30 mL) and dried. Further drying in an oven at 40°C. under high vacuum for 18 h provided 12.4 g of a solid product(quantitative yield).

1,1,2-Trimethylbenz[e]indole-6,8-disulfonate (2)

A solution of 6-hydrazino-1,3-naphthalenedisulfonate (10.3 g, 32.5mmol), isopropylmethylketone (7.6 mL, 71 mmol), potassium acetate (6.82g, 69 mmol) in acetic acid (50 mL) was heated under reflux in an oilbath for 24 h. After cooling to ambient temperature the solvent wasevaporated off under reduced pressure to dryness and triturated withiPrOH (100 mL). The resultant solid was collected, washed with iPrOH(2×30 mL) and dried. Further drying in an oven at 40° C. under highvacuum for 18 h provided the solid product (9.59 g, 80%).

1,1,2-Trimethyl-3-(3-sulfopropyl)benz[e]indolium-6,8-disulfonate (3)

A suspension of 1,1,2-trimethylbenz[e]indole-6,8-disulfonate (2.5 g, 6.8mmol) and 1,3-propanesultone (2.16 mL, 24.4 mmol) in 1,2-dichlorobenzene(50 mL) was heated in an oil bath at 140° C. for 24 h. After cooling toambient temperature the solvent was decanted, the solid was washed withethyl acetate (3×20 mL) and solvent was decanted. The solid wastriturated with methanol (50 mL) and then ethyl acetate (50 mL) wasadded. Filtered to collect the solid, washed with ethyl acetate (2×20mL), ether (2×20 mL) and dried in an oven at 40° C. under high vacuumfor 18 h to afford 1.9 g (50%) of a solid product.

3-Carboxypentyl-1,1,2-Trimethylbenz[e]indolium-6,8-disulfonate (4)

A suspension of 1,1,2-trimethylbenz[e]indol-6,8-disulfonate (4.82 g,13.0 mmol), bromohexanoic acid (25 g, 130 mmol) in a 250 mL round bottomflask was heated in an oil bath at 125° C. for 40 h. After cooling toambient temperature ethyl acetate (100 mL) was added the solid wastriturated. Filtered to collect the solid, washed with ethyl acetate(3×50 mL), ether (2×20 mL) and dried. Further drying in an oven at 40°C. under high vacuum for 18 h gave 4.41 g (70.0%) of the product.

2-[(1E,3E)-4-Anilinobuta-1,3-dienyl]-1,1-Dimethyl-3-(3-sulfopropyl)benz[e]indolium-6,8-disulfonate(5)

A solution of1,1,2-trimethyl-3-(3-sulfopropyl)benz[e]indolium-6,8-disulfonate (1.03g, 2.10 mmol) and malonaldehyde dianil hydrochloride (596 mg, 2.30 mmol)in acetic acid (10 mL) was heated to reflux for 18 h. The progress ofthe reaction was monitored with analytical HPLC for the disappearance ofthe starting material, and the formation of the product. Solvent wasremoved under reduced pressure and the residual dark solid was washedwith ethyl acetate (3×20 mL). The dried solid was used without furtherpurification in the next reaction for dicarbocyanine dye synthesis.

Preparation of 6

To a solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-1,1-Dimethyl-3-(3-sulfopropyl)benz[e]indolium-6,8-disulfonate(69.9 mg, 0.114 mmol),3-carboxypentyl-1,1,2-Trimethylbenz[e]indolium-6,8-disulfonate (55.2 mg,0.114 umol) in N,N-dimethylformamide (1.0 mL) was added acetic anhydride(200 uL) and triethylamine (200 uL) and stirred at ambient temperaturefor 18 h. Solvent was evaporated off under reduced pressure to give adark blue residue, which was then purified by reverse-phase HPLC(acetonitrile/0.1 M TEAB gradient) to give the product 6 (λmax 677 nm).

Preparation of dCTP-6C-Lys(Boc)-NH₂ (7)

To a solution of Fmoc-D-Lys(Boc)-OPfp (4.5 mg, 7.09 umol) in DMF (400uL) was added a solution of dCTP-6C—NH₂ (3 umol) in 0.1 M NaHCO₃, pH 8.3aqueous buffer (200 uL) at ambient temperature for 18 h. To the solutionwas then added triethylamine (500 uL) and stirred for 5 h. Solvent wasevaporated off under reduced pressure to give a solid, which waspurified by reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) togive 2.05 umol of the desired product (68.3% yield).

Preparation of dCTP-6C-Lys(Boc)-Lys(Boc)-NH₂ (8)

To a solution of Fmoc-D-Lys(Boc)-OPfp (1.65 mg, 2.60 umol) in DMF (200uL) was added a solution of dCTP-6C-Lys(Boc)-NH₂ (1.30 umol) in 0.1 MNaHCO₃, pH 8.3 aqueous buffer (100 uL) at ambient temperature for 18 h.To the solution was then added triethylamine (400 uL) and stirred for 5h. Solvent was evaporated off under reduced pressure to give a solid,which was purified by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) to give 0.75 umol of the desired product (58% yield).

Preparation of dCTP-6C-Lys(Boc)-Lys(Boc)-Lys(Boc)-NH₂ (9)

To a solution of Fmoc-D-Lys(Boc)-OPfp (1.80 mg, 2.80 umol) in DMF (200uL) was added a solution of dCTP-6C-Lys(Boc)-Lys(Boc)-NH₂ (0.50 umol) in0.1 M NaHCO₃, pH 8.3 aqueous buffer (100 uL) at ambient temperature for18 h. To the solution was then added triethylamine (400 uL) and stirredfor 5 h. Solvent was evaporated off under reduced pressure to give asolid, which was then purified by reverse-phase HPLC (acetonitrile/0.1 MTEAB gradient) to give the desired product.

Preparation of 10

To a solution of the activated ester of dicarbocyanine dye (1 umol) inDMF (100 uL) in an Eppendorf tube was added a solution ofdCTP-6C-Lys(Boc)-NH₂ (0.6 umol) in 0.1 M NaHCO₃, pH 8.3 aqueous buffer(100 uL) at 0° C. After brief vortexing the solution was let stood for18 h in the dark at ambient temperature. The solution was then subjectedto reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) purification togive the adduct, which was then treated with 3 M HCl (600 uL) at ambienttemperature for 1 h. The solution was again subjected to reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) purification to give the product(0.43 umol, 71% yield).

Preparation of 11

To a solution of the activated ester of dicarbocyanine dye GS290-103 (1umol) in DMF (100 uL) in an Eppendorf tube was added a solution ofdCTP-6C-Lys(Boc)-Lys(Boc)-NH₂ (0.25 umol) in 0.1 M NaHCO₃, pH 8.3aqueous buffer (100 uL) at 0° C. After brief vortexing the solution waslet stood for 18 h in the dark at ambient temperature. The solution wasthen subjected to reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient)purification to give the adduct, which was then treated with 1.5 M HCl(1.2 mL) at ambient temperature for 18 h. The solution was againsubjected to reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient)purification to give the product (0.12 umol, 47% yield).

Preparation of 12

To a solution of the activated ester of dicarbocyanine dye (1 umol) inDMF (100 uL) in an Eppendorf tube was added a solution ofdCTP-6C-Lys(Boc)-Lys(Boc)-Lys(Boc)-NH₂ (0.1 umol) in 0.1 M NaHCO₃, pH8.3 aqueous buffer (100 uL) at 0° C. After brief vortexing the solutionwas let stood for 18 h in the dark at ambient temperature. The solutionwas then subjected to reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) purification to give the adduct, which was then treated with 3M HCl (0.2 mL) at ambient temperature for 18 h. The solution was againsubjected to reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient)purification to give the product (0.02 umol, 20% yield).

Preparation of dCTP-6C-Glu(Boc)-NH₂ (13)

To a solution of Fmoc-D-Glu(O-tBu)-OPfp (6 umol) in DMF (400 uL) wasadded a solution of dCTP-6C—NH₂ (4 umol) in 0.1 M NaHCO₃, pH 8.3 aqueousbuffer (200 uL) at ambient temperature for 18 h. To the solution wasthen added triethylamine (400 uL) and stirred for 18 h. Solvent wasevaporated off under reduced pressure to give a solid, which waspurified by reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) togive 2.8 umol of the desired product (70% yield).

Preparation of dCTP-6C-Glu(O-tBu)-Glu(O-tBu)-NH₂ (14)

To a solution of Fmoc-D-Glu(O-tBu)-OPfp (6 umol) in DMF (200 uL) wasadded a solution of dCTP-6C-Glu(O-tBu)-NH₂ (2.4 umol) in 0.1 M NaHCO₃,pH 8.3 aqueous buffer (100 uL) at ambient temperature for 18 h. To thesolution was then added triethylamine (400 uL) and stirred for 4 h.Solvent was evaporated off under reduced pressure to give a solid, whichwas purified by reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) togive 1.42 umol of the desired product (58% yield).

Preparation of dCTP-6C-Glu(O-tBu)-Glu(O-tBu)-Glu(O-tBu)-NH₂ (15)

To a solution of Fmoc-D-Glu(O-tBu)-OPfp (4 umol) in DMF (200 uL) wasadded a solution of dCTP-6C-Glu(O-tBu)-Glu(O-tBu)-NH₂ (1.0 umol) in 0.1M NaHCO₃, pH 8.3 aqueous buffer (100 uL) at ambient temperature for 18h. To the solution was then added triethylamine (400 uL) and stirred for18 h. Solvent was evaporated off under reduced pressure to give a solid,which was then purified by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) to give the desired product.

Preparation of 16

To a solution of the activated ester of dicarbocyanine dye (1 umol) inDMF (100 uL) in an Eppendorf tube was added a solution ofdCTP-6C-Glu(O-tBu)-NH₂ (1.0 umol) in 0.1 M NaHCO₃, pH 8.3 aqueous buffer(100 uL) at 0° C. After brief vortexing the solution was let stood for18 h in the dark at ambient temperature. The solution was then subjectedto reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) purification togive the adduct, which was then treated with 3 M HCl (600 uL) at ambienttemperature for 18 h. The solution was again subjected to reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) purification to give the product(0.14 umol, 14% yield).

Preparation of 17

To a solution of the activated ester of dicarbocyanine dye (1 umol) inDMF (100 uL) in an Eppendorf tube was added a solution ofdCTP-6C-Glu(O-tBu)-Glu(O-tBu)-NH₂ (0.5 umol) in 0.1 M NaHCO₃, pH 8.3aqueous buffer (100 uL) at 0° C. After brief vortexing the solution waslet stood for 18 h in the dark at ambient temperature. The solution wasthen subjected to reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient)purification to give the adduct, which was then treated with 3 M HCl(0.4 mL) at ambient temperature for 18 h. The solution was againsubjected to reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient)purification to give the product (0.21 umol, 41% yield).

Preparation of 18

To a solution of the activated ester of dicarbocyanine dye (1 umol) inDMF (100 uL) in an Eppendorf tube was added a solution ofdCTP-6C-Glu(O-tBu)-Glu(O-tBu)-Glu(O-tBu)-NH₂ (1 umol) in 0.1 M NaHCO₃,pH 8.3 aqueous buffer (100 uL) at 0° C. After brief vortexing thesolution was let stood for 18 h in the dark at ambient temperature. Thesolution was then subjected to reverse-phase HPLC (acetonitrile/0.1 MTEAB gradient) purification to give the adduct, which was then treatedwith 3 M HCl (0.2 mL) at ambient temperature for 18 h. The solution wasagain subjected to reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient)purification to give the product (0.18 umol, 18% yield).

The following examples set forth the preparation of sulfocystinelinkers.

Preparation of Alexa555-SC (28)

To a solution of sulfocysteine (60 mg, 320 umol) in DMF (1.4 mL) wasadded a solution of Alexa555-NHS (16 mg, 13 umol) in DMF (1.0 mL),followed by addition of DIPEA (0.80 mL). The solution was stirred atroom temperature in the dark for 18 h. After concentrating to drynessthe residue was subjected to reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) purification to give the product (8.68 umol, 67% yield).

Preparation of Alexa555-SC-NHS (29)

To a solution of Alexa555-SC (2.0 umol) in DMF (200 uL) in an Eppendorfvial was added carbonyl diimidazole (CDI, 5 mg, excess) followed byN-hydroxysuccimide (NHS, 5 mg, excess) and vortexed. After standing inthe dark for 18 h added ethyl acetate (1.3 mL) and vortexed.Centrifugation of the vial at high speed gave a pellet of product, andthe solvent was decanted. The pellet was dried to give a crude productof the activated NHS ester. The product was used in the couplingreaction without further purification.

Preparation of Alexa555-SC-SC (30)

To a solution of sulfocysteine (10 mg, 53 umol) in 0.1 M NaHCO₃, pH 8.5(200 uL) was added a solution of Alexa555-SC-NHS (2.0 umol) in DMF (200uL). The solution was stood at room temperature in the dark for 18 h.After concentrating to dryness the residue was subjected to ion-exchangeseparation (1 M TEAB/0.05 TEAB) followed by reverse-phase HPLC(acetonitrile/0.1 M TEAB gradient) purification to give the product(1.38 umol, 69% yield).

Preparation of Alexa555-SC-SC-NHS (31)

To a solution of Alexa555-SC-SC (1.38 umol) in DMF (100 uL) in anEppendorf vial was added carbonyl diimidazole (CDI, 5 mg, excess)followed by N-hydroxysuccimide (NHS, 5 mg, excess) and vortexed. Afterstanding in the dark for 18 h added ethyl acetate (1.3 mL) and vortexed.Centrifugation of the vial at high speed gave a pellet of product, andthe solvent was decanted. The pellet was dried to give a crude productof the activated NHS ester. The product was used in the couplingreaction without further purification.

Preparation of Alexa555-SC-6C-dT6P (32)

To a solution of the activated ester of Alexa-SC-NHS (1 umol) in DMF(100 uL) in an Eppendorf tube was added a solution of dT6P-6C—NH₂ (1umol) in 0.1 M NaHCO₃, pH 8.3 aqueous buffer (100 uL) at 0° C. Afterbrief vortexing the solution was let stood for 18 h in the dark atambient temperature. The solution was then subjected to ion-exchangeseparation (1 M TEAB/0.05 TEAB) followed by reverse-phase HPLC(acetonitrile/0.1 M TEAB gradient) purification to give the product(0.12 umol, 12% yield).

Preparation of Alexa555-SC-6C-dT6P (33)

To a solution of the activated ester of Alexa-SC-SC-NHS (1.38 umol) inDMF (100 uL) in an Eppendorf tube was added a solution of dT6P-6C—NH₂(1.5 umol) in 0.1 M NaHCO₃, pH 8.3 aqueous buffer (100 uL) at 0° C.After brief vortexing the solution was let stood for 18 h in the dark atambient temperature. The solution was then subjected to ion-exchangeseparation (1 M TEAB/0.05 TEAB) followed by reverse-phase HPLC(acetonitrile/0.1 M TEAB gradient) purification to give the product(0.33 umol, 24% yield).

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

All patents, patent applications, and other publications cited in thisapplication are incorporated by reference in the entirety.

1.-14. (canceled)
 15. A fluorescent dye having the formula:{R¹-(L¹)_(a)-(AA)_(n)}_(y)-(L²)_(b)-X wherein R¹ is a fluorescent dyemoiety; (AA)_(n) is an amino acid linker; n is a selected from theintegers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and when n is two orgreater, each n amino acid is independently selected; X is a polyvalentmoiety, comprising a moiety having the structure:

wherein Y is a nucleobase; and u is selected from the integers 1, 2, 3,4, 5, 6, 7 and 8; y is selected from the integers 1, 2, 3, 4, 5, 6, 7and 8, such that when y is 2 or greater, X is a polyvalent moiety; L¹and L² are selected from bonds and adaptors; a is selected from theintegers 0 and 1; and b is an integer selected from 0, 1, 2, 3, 4, 5, 6,7 and
 8. 16. The fluorescent dye according to claim 15 wherein saidpolyvalent moiety comprises bonded thereto a member selected from asecond fluorescent dye moiety, a nucleic acid and a combination thereof.17. The fluorescent dye according to claim 16, wherein said firstfluorescent dye moiety and said second fluorescent dye moiety form aFRET pair.
 18. The fluorescent dye of claim 15, wherein said amino acidlinker includes amino acids selected from hydrophobic amino acids,cationic amino acids, and a combination thereof.
 19. The fluorescent dyeof claim 18, wherein said amino acid linker further comprises an anionicamino acid.
 20. The fluorescent dye of claim 15, wherein n is 1, 2, 3,4, 5, or
 6. 21. The fluorescent dye of claim 15, wherein the amino acidsforming (AA)_(n) are the same amino acid or a different amino acid. 22.The fluorescent dye of claim 15, wherein said amino acid linker is otherthan Pro, Pro₂, Pro₃, Pro₄, Pro₅ or Pro₆.
 23. The fluorescent dye ofclaim 15, wherein said amino acid linker is other than Lys, Lys₄, Lys₅or Lys₆.
 24. The fluorescent dye of claim 19, wherein said anionic aminoacid is a member selected from, sulfocysteine, glutamic acid, andO-phosphoserine.
 25. The fluorescent dye of claim 15, wherein L¹ and L²independently comprise an adaptor selected from an alkyl amine and anitrogen-containing heterocyclic moiety.
 26. The fluorescent dye ofclaim 25, wherein said alkyl amine is:—NH(CH₂)_(g)C(O)NH(CH₂)_(h)—, wherein g and h are independently selectedfrom the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher.
 27. Thefluorescent dye of claim 15, wherein each of said n amino acids is thesame amino acid.
 28. The fluorescent dye of claim 15, wherein saidfluorescent dye moiety is a cyanine dye.
 29. The fluorescent dye ofclaim 28, wherein said cyanine is selected from Cy3 and Cy5 cyaninedyes.
 30. The fluorescent dye of claim 15, wherein said polyvalentmoiety is a residue of a member selected from triazine, perylene,piperidine, phenylalanine, diaminopropanoic acid, aspartic acid, lysine,glutamic acid, serine, aminoadipic acid, 3,5-dihydroxybenzoic acid,2-amino-4-hydroxy-butyric acid, 4-(1-amino-1-carboxyethyl)-benzoic acid,piperazine-2-carboxylic acid,4-[4,6-bis-(piperidin-4-ylamino)-[1,3,5]triazin-2-ylamino]-cyclohexanecarboxylicacid and 3-amino-3-[4-(3-amino-prop-1-ynyl)-phenyl]-propionic acid. 31.The fluorescent dye of claim 15, wherein said fluorescent dye binds to aDNA polymerase with a binding constant greater than an otherwiseidentical fluorescent dye in which (AA)_(n) is absent.
 32. Thefluorescent dye of claim 15 wherein said amino acid comprises a sidechain comprising a moiety having the formula:

in which each R is independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl.
 33. Amethod of monitoring an enzyme reaction, said method comprising: (a)forming a reaction mixture by contacting said enzyme with a fluorescentdye according to claim 15, wherein said dye is a substrate for saidenzyme under conditions sufficient for said enzyme and said dye toreact; and (b) monitoring fluorescence of said reaction mixture.
 34. Themethod according to claim 33, wherein said enzyme is a DNA polymeraseand said dye comprises a nucleic acid moiety which is said substrate forsaid enzyme.
 35. The method according to claim 33 wherein said enzymereaction is template directed DNA synthesis.
 36. The method according toclaim 35, wherein said reaction is a component of a single molecule DNAsequencing analysis.